Heat-stable enzyme and its use

[Detailed Description of the Invention]

The [industrial use field] The present invention relates to refined thermostable enzyme. In one mode, this enzyme is the DNA polymerase refined from Thermes Aquaticus (Thermus a uaticus), and that molecular weight is about 86.000~90.000. The present invention will amplify them, if the existing nucleic acid arrangement exists in a test sample, and supposing it exists, it relates to the method of detecting them by using a probe. The present invention makes easy detection of the arrangement of production Flip to this and others in large quantities as compared with the quantity which exists specific nucleic acid arrangement first from the predetermined arrangement of DNA or RNA, and, specifically, relates to the method of using the thermostable enzyme which carries out the catalyst of the reaction. DNA or RNA may be a chain or 2 chains in the end of -, and the method of the 0 present invention which may be one ingredient of the mixture of a comparatively pure kind or nucleic acid uses a repetitive reaction in order to attain amplification of nucleic acid arrangement [ Desire ]. 0 to which extensive research has been done about isolation of the DNA polymerase from [Description of the Prior Art] E and a moderate temperature microorganism like Cori (E, coli) -- for exampleBeth Mann CBessman et al., J, Biol, and Chem (1957), Please refer to 233 volumes, 171~177 pages and Butyne (13uttin), Korn berg (Korn-berg) (1966) J, Biol, and Chem, 241 volumes, and 5419~5427 pages. By contrast, research is seldom done about isolation and refining of the DNA polymerase from thermophilic bacteria like Thermes Aquaticus CThermus air. Kalesin et al. (Kaledin), 7* (1980) 45 volumes, and 644~651 pages of six-step isolation and the refining processes of the DNA polymerase from the cell of a Thermes Aquaticus (T, victory n A time cus) YTI stock are indicated. These processes comprise the judgment on isolation of crude extracts, DEAE-cellulose chromatography, and a hydroxy apatite, the judgment on DEAE-cellulose, and the chromatography on vehicle-ffDNA-cellulose. It has been reported per monomer unit that the molecular weight of the refined enzyme by which the pool from each stage is not screened about mixing of an end and Exorso nuclease is 62.000 Dalton. Thermes Aquaticus (the second refining process about 7 and 7 Calah (7) polymerase is indicated by 127 volumes and 1550~1557 pages in A Choi 7 (A, Chien) et al., J, Bacteriol, and (1976).) In this method, crude extracts are covered over a DEAE- Sephadex column. Next, pool-under dialysis part drawing is given to the processing on a phospho cellulose column. this Poole -- a part for the bottom -- drawing -- it dialyzes, and bovine serum albumin (BSA) is added and the active loss of polymerase is prevented. The mixture to generate is applied to a DNA-cellulose column. it pooled from the column -- when substance dialysis is carried out and gel filtration analyzes, a molecular weight is about 63 and 000 Dalton, and, according to sucrose centrifugal separation, are about 68.000 Dalton. The method of amplifying the existing nucleic acid arrangement in large quantities compared with the quantity which exists first is suggested to the European patent public presentation No. 200, 362 specification of December 10, 1986 issue using thermostable enzyme. In this method, primer nucleotide triphosphate and polymerase are used and it consists of composition and the hybridization of denaturation and molds. The extension output of each primer becomes a mold for manufacturing desired nucleic acid arrangement. In this specification, since that activity is not destroyed by heat when the polymerase used is thermostable enzyme, it is not necessary to add it after each denaturation process. About use of the refined heat stability DNA polymerase, Also the amplification and the detection process which are not provided at all about other profits and detailed points, It is indicated to 5cience of Psyche and others (Saiki), 230 volumes, 1350~1354 pages (1985), -Bio Tech- Dogei of Psyche and others (Saiki), three volumes, and 1008~1012 pages (1985). [Problem (s) to be Solved by the Invention] Therefore, in this industry, to manufacture the refined stable thermostable enzyme which can be used in order to improve the above-mentioned amplification process for diagnosis is desired. [Means for Solving the Problem] Therefore, the present invention provides refined thermostable enzyme which carries out the catalyst of the combination of nucleotide triphosphate, and forms complementary nucleic acid in nucleic acid molds. Preferably, refined enzyme is Thermes Aqua J" A (it is the DNA polymerase of the Thermus 7 origin, and a molecular weight is about 86, 000~90, 000 Dalton.). Use this refined material for a temperature cycle amplification reaction, nucleic acid arrangement is made to produce in large quantities from predetermined nucleic acid arrangement as compared with quantity which exists first, and it can make it possible to detect them easily. Thermes Aquaticus (a gene which codes enzyme from DNA polymerase of Thermus Bowed Dish origin is also identified, and another means to collect thermostable enzyme of the present invention is provided.) In addition to a gene which codes enzyme of about 86 and 000~90.000 Dalton, a genetic induction object which codes DNA polymerase activity is also provided. Finally, the present invention also includes a stable enzyme constituent which contains the refined above thermostable enzyme in buffer solution containing one or more sorts of nonionic polymer detergent. Enzyme produced by refined enzyme and recombinant DNA art provides singularity far higher than the Freneau (Klenow) fragmentation which is not heat stability. Enzyme produced by refined enzyme and recombination art shows suitable activity expected when dTTP or other nucleotide triphosphate does not exist in an in Kuvesigon mixture with a DNA mold. These enzyme consists a Thermes Aquaticus (J larger than pH of thermostable enzyme of Thermus Daisuke Tamashi origin ) H range indicated in literature, In pH 7, these thermostable enzyme can be stability further, without saving in buffer solution in which the bigger activity of a case in pH 8 than 50% is shown and which has nonionic detergent, and losing activity as Mutually in a long time. The present invention is in a method of amplifying one sort or two or more sorts of specific nucleic acid arrangement which exists in nucleic acid or those mixtures using a primer and thermostable enzyme. If extension output of one primer hybridizes to other things, it becomes a mold of a production To shake sake about desired specific nucleic acid arrangement, and that contrary will also be the same and this method will be repeated only the number of times required for that of production To shake in arrangement of a desired quantity. This method raises the singularity of an amplification reaction and produces a very clear signal of amplified nucleic acid. A method of the present invention abolishes the necessity of moving a reagent from one container after each amplification cycle at another container. Since thermostable enzyme has tolerance to high temperature which takes a nucleic acid chain to denature and does not so need to exchange it, the above moving is not required. Temperature cycle change can be automated and the number of processes can further be reduced with a staff required to perform an amplification reaction. When the present invention is the method of amplifying at least one sort of specific nucleic acid arrangement included in a mixture of nucleic acid or nucleic acid and nucleic acid is specifically 2 chains, This nucleic acid consists of two separate complementary chains of length which is the same or different, (a) Contact each nucleic acid chain to one oligonucleotide primer about four different nucleotide triphosphate and each different specific arrangement which should be amplified, However, when extension output which each primer was substantially chosen as a chain with which each specific arrangement differs complementarily, and was compounded from one primer dissociates from the phase assistant object, It enables it to work as a mold of composition of extension output of other primers. performing the above-mentioned contact at temperature which promotes a hybridization to the complementary nucleic acid chain of each primer --; (b) -- each nucleic acid chain, Thermostable enzyme which carries out the catalyst of the combination of nucleotide triphosphate is made to contact after this process simultaneously with a process (a). About each different arrangement which a complementary primer extension output is formed in each chain of each nucleic acid, and; (c) enzyme is activated, and should be amplified, In temperature which is not high temperature to the extent that it is an effective temperature in order to compound extension output of each complementary primer to each nucleic acid chain mold, but each extension output is separated from the complementary chain mold, It continues for effective time, In temperature which is not high temperature to the extent that it is an effective temperature in order to separate the primer extension output from a mold which held a mixture from a process (b), and in which : (d) primer extension output was compounded on it and to make 1 chain molecule generate, but enzyme is denatured irreversibly, It continues for effective time, a mixture from a process (c) is heated, and it is a mixture from; (e) process (d), In order to promote a hybridization through which 1 chain molecule produced at a process (d) of each primer passes, respectively, it cools over time effective in an effective temperature, and promotes the activity of; (f) enzyme, And about each different arrangement which should be amplified, In order to compound extension output of each complementary primer to each nucleic acid chain mold produced at a process (d), it is an effective temperature, but each extension output is continued for effective time at temperature which is not high temperature to the extent that it dissociates from the complementary chain mold, A mixture from a process (b) is held. A method of coding performing process (e) and (f) being simultaneous or one by one is provided. Process (d), (e), and (f) is repeatable until amplification of arrangement of a level [ Desire ] is obtained. Desirable thermostable enzyme given [this] in a specification is Thermes Aquaticus (it is the polymerase (Taq polymerase) by which T h e r m u s 7 Calah extraction was carried out most preferably). When enzyme is Taq polymerase, at a process (a), it is a nucleic acid chain, Aid solution containing each nucleotide of magnesium salt of abbreviation 1.5~2mM and 150~200 enclosure and each primer of 1 Oh is made to contact, process (a), (e), and (f) is performed by about 45~58 degreeC, and a process (d) is performed by about 90~100 degreeC. In a desirable mode, one sort or two or more sorts of nucleic acid is 2 chains, and a process (a) is, (i) each nucleic acid -- four different nucleotide triphosphate -- and -- each different specific arrangement which should be amplified -- the bottom of one existence of an oligonucleotide primer, Each nucleic acid is heated to time effective in denaturing, and an effective temperature, However, when extension output which each primer was substantially chosen as a chain with which each specific arrangement differs complementarily, and was compounded from one primer dissociates from the phase assistant object, It is carried out by enabling it to work as a mold of composition of extension output of other primers, and cooling nucleic acid which (ii) -denatured to temperature which promotes a hybridization to the complementary nucleic acid chain of each primer. In other modes, the present invention detects existence of at least one sort of specific nucleic acid arrangement, or absence in a sample containing a mixture of nucleic acid or nucleic acid, Or when it is the method of identifying two sorts in the above-mentioned sample of different arrangement, and a sample is expected including the above-mentioned l or a plurality of arrangement and 1 or a plurality of nucleic acid are 2 chains, They consist of two separated complementary chains of length which is not equal or equal, respectively, process (a) (f) ~ is the same as the above, and it is one specific nucleic acid arrangement. Or if it exists, quantity of a plurality of arrangement will be made to amplify, and process (e) and (f) will be performed being simultaneous or one by one, (g) To output of a process (f), it is in the above-mentioned arrangement, or can hybridize to the mutant, It is related with a method of coding determining whether added an oligonucleotide probe which carried out the sign for each arrangement which should be detected, and; and the (h) above-mentioned hybridization happened. In another 641, the present invention is the method of detecting existence of change of at least one nucleotide sequence or absence in one sort contained in a sample, or two or more sorts of nucleic acid, Only number of times sufficient when it consists of two separated complementary chains of length which is not equal or equal, respectively when nucleic acid is 2 chains, and ■ or a plurality of arrangement change exist including the above-mentioned process (a) (f) ~ to obtain amplification which can detect nucleic acid containing these is a process (d). (e) And (f) is repeated and process (e) and (f) is performed being simultaneous or one by one, (g) Output of a process (f) can be fixed to a film and it can hybridize with nucleic acid arrangement amplified only when complementary to a field of arrangement to which arrangement of the (h) probe was amplified, It is related with a method of processing the above-mentioned film under hybridization conditions, and coding that; and a (i) probe detect whether it hybridized in arrangement by which it was amplified in a nucleic acid sample with an arrangement specific oligonucleotide probe by which the sign was carried out. When a sample contains a cell, preferably, those cells are heated before a process (a) and exposed to a Maki talk reagent in it. This process avoids extraction of nucleic acid before addition of a reagent. It is made to carry out the sign of the amplified arrangement which carries out the sign of one sort or a plurality of primers, and/or the nucleotide phosphate, and generates them in a strange method of this method. One sort or a plurality of primers, and/or nucleotide phosphate by which the sign was carried out may exist in a reaction mixture from the beginning, or it can also add them in a next cycle. (Non-sign) It fixes to a film and arrangement specific oligonucleotide triphosphate is processed under hybridization conditions by amplification output by which the sign was carried out, and it is made for a hybridization to happen only when arrangement combined with a film exists in amplification output. In another mode, the present invention is the method of clone-izing one sort or two or more sorts of specific nucleic acid arrangement included in a mixture of nucleic acid or nucleic acid to a cloning vector, When one sort or a plurality of nucleic acid are 2 chains, two separated complementary chains, The one sort or two or more sort nucleic acid is quantitatively amplified in front of cloning, and this method includes the above-mentioned process (a) (f) ~, Only sufficient number of times for a process (d), Ce", and (f) to produce amplification which can detect one sort including one sort or two or more sorts of arrangement or two or more sorts of nucleic acid adds a restriction enzyme to each of the above-mentioned restriction site to output of; (g) process (f) repeatedly, A process of including specific arrangement which obtains cleavage output and should be formed into; (h) clone in a restriction digestive thing (related with a method of consisting of connecting cleavage output of g> with one or more cloning vectors.) In the last mode, the present invention is the method of clone-izing one sort or two or more sorts of specific nucleic acid arrangement included in a mixture of nucleic acid or nucleic acid to a cloning vector, It consists of two separated complementary chains which are not [that length is equal when one sort or two or more sorts of nucleic acid is 2 chains, or] equal, the one sort or two or more sorts of nucleic acid is quantitatively amplified in front of cloning, this method includes the above-mentioned process (a) (f) ~, and it is a process (d). (e) And in order to carry out flush end connection of the (f) at one or more sorts of cloning vectors, Only sufficient number of times to produce one sort including one sort or a plurality of arrangement or amplification which can detect a plurality of nucleic acid is repeated, (g) Connect one sort which was obtained from a process (f) and which should be clone-ized, or two or more sorts of amplified specific sequences with one sort or two or more sorts of above-mentioned cloning vectors under existence of ligase, and it is here, It is related with a method of coding existing in sufficient quantity for one sort or two or more sorts of arrangement amplified [above-mentioned] and one sort, or two or more sorts of vectors to connect. In a mode of output, the present invention is a constituent useful although at least one sort of specific nucleic acid arrangement included in a mixture of nucleic acid or nucleic acid is amplified, About each different specific arrangement which should be amplified, four different nucleotide triphosphate and one oligonucleotide primer are contained, Each primer can be substantially chosen as a chain with which each specific arrangement differs complementarily, and extension output compounded from one primer can be committed now as a mold of composition of extension output of other primers by having dissociated from the phase assistant object. In a mode of another output, the present invention provides a sample of one sort or two or more sorts of nucleic acid which is included in nucleic acid and containing two or more products of specific nucleic acid arrangement. This sample can have a chain of about 10~100, a chain of about 100~1000, or a chain exceeding about 1000. In a mode of the last output, the present invention provides nucleic acid arrangement amplified from a mixture of nucleic acid containing a plurality of copies of arrangement produced by a method of amplifying the present invention, or nucleic acid. "cell" "cell line (cellular in) J and a "cell culture thing" which are used for this specification can be used so that mutual exchange is possible, and all of these names include posterity. Therefore, a culture which originates without a relation to - next cell and the number of times of transfer in the cell is included with a "transformant" or "a transformed cell. "It is also understood that all the posterity has that a DNA content is not correctly the same by intentionally or accidental mutation. Mutant posterity who has the same function as having placed and screened into a cell transformed first is also included. The term "control arrangement" points out NA arrangement to revelation of a coding sequence connected so that an operation in a specific host living thing was possible in necessity. The suitable control arrangement for a procaryote can also include a promotor and arrangement others are seldom understood to be although there are operator arrangement and a ribosome binding site arbitrarily, for example. It is known that a eucaryotic cell will use a promotor, the Pori Ade Nils-ized signal, and an enhancer. protein in which a host who the term a "revelation system" points out DNA arrangement including a desired coding sequence and control arrangement which were connected so that an operation was possible, and is transformed in these arrangement was coded -- production To shake -- things are made. In order to transform, a vector may be made to include a revelation system, but [next] DNA of relation may be included in a host chromosome. The term a "gene" used on these specifications points out DNA arrangement which codes polypeptide or a precursor which has callable biological activity. gene sequences of length with this perfect polypeptide -- or it may be coded by one portion of the coding sequences as long as enzyme activity is held. A term of "having connected so that an operation was possible" points out juxtaposition which can perform a normal function of an ingredient. For example, a coding sequence "was connected so that an operation was possible" with control arrangement points out arrangement which this coding sequence can reveal under control of control arrangement. "Nonionic polymer detergent" does not have an ionicity electric charge, but points out a surface-active agent which codes that enzyme can be stabilized the pif range of abbreviation 3.5~abbreviation 9.5, preferably in [pl] 4~8.5 to the object of the present invention. The term an "oligonucleotide" used for this specification is defined as a molecule which consists of two or more deoxyribonucleotides and beam Polynucleotide , preferably three or more things. The exact size changes with many factors, and these factors change with final functions or uses of an oligonucleotide. an oligonucleotide is composition -- it can guide by cloning-like. The term a "primer" used for this specification, It is an oligonucleotide in a generated restriction digestive thing manufactured in composition so that may recognize natural existence, In inside of conditions which induce composition of a complementary primer extension output to a nucleic acid chain, i. e., suitable buffer solution, (pH, ionic strength, a cofactor, etc. are included with "buffer solution"), When placing at a suitable temperature under four sorts of conditions of existence of different nucleotide triphosphate and thermostable enzyme, what can act as the composite starting point is pointed out. About Taq polymerase, it is buffer solution of this specification, containing each nucleotide of magnesium salt of 1.5~2mM preferably MaCj!z, and a 150~200 team, and each primer of 1 Prostitution preferably -- desirableTris buffer solution (pH 8~8.4) of KCl of 501IM and 10mM and gelatin of 1100JI/- are included. Although this primer is a single chain preferably in amplification to acquire the maximum efficiency, 2 chains may be sufficient as it. In the case of 2 chains, a primer is processed first, and before using for preparing extension output, it is separated for every chains of those. Preferably, a primer is oligo deoxyribonucleotide. A primer must be long enough and must be able to carry out the prime of the composition of extension output in existence of thermostable enzyme. The exact length of a primer changes by a factor of many like temperature, the primer origin, and a method use. For example, although an oligonucleotide primer contains 15~25 nucleotide typically depending on complexity of target arrangement, from it, it is many or nucleotides fewer than it may be included. In order to form a mold and a hybrid complex stable enough, generally, low temperature is required for a short primer molecule. A chain with which each specific sequences by which these primers are amplified differ "it is chosen so that it may become a J phase assistant target substantially. This means having to be sufficiently complementary for carrying out hybrid formation with each of those chain. So, the primer arrangement does not need to reflect exact arrangement of a mold. for example, -- combine un-complementary nucleotide fragmentation with 5' end of a primer, and the remaining primer arrangement is complementary to a chain -- making. When becoming a mold for composition of extension output of other primers by being complementary enough and carrying out hybrid formation with this chain to arrangement of a chain with which primer arrangement is amplified, a non-complementary base or longer arrangement can be included in a primer. However, in order to detect using an arrangement specific probe which carried out the sign in detail, the highest result is obtained by the object of detection when a primer has typically exact complementarity. A term called [restrictions, nuclease", and a "restriction enzyme" which are used for this specification is bacterial enzyme, and points out that from which each cuts double iJ D N A of a specific nucleotide sequence or its neighborhood. A term called rDNA many forms where it is used for this specification" points out the state where two or more sorts of different nucleotide sequences can exist in a specific part of DNA. The term "change of a nucleotide sequence" used for this specification points out nucleotide substitution single or multiple [a certain kind of], deficiency, or insertion. These nucleotide change may be mutation or a polymorphism allelic variation. So, beta-globulin Inheritance from which a method used for this specification is started by variation of a single base, addition, or deficiency (beta- Thalassemia of a certain kind) A large number base variation which can also detect single nucleotide change by nucleic acid which happens in Fiber -like red corpuscle anemia, hemoglobin C Disease , etc., and is included by alpha- Thalassemia or beta- Thalassemia of a certain kind is also detectable. A part with specific nucleic acid in a group in which a method of the present invention is not related inevitably sick, For example, many forms which are in a state where two (it is (like a replaced nucleotide base pair which deficiency was carried out or was inserted)) or more sorts of different nucleotide sequences exist in a flower at a human genome and random many forms, for example, HL A63 region of mitochondrial DNA, are detectable. An oligonucleotide probe specific in multi-form arrangement indicated in detail below is used for detecting a hereditary marker related sick like diabetes Meritus (mellitus) of insulin dependence, or can also be used for application of forensic medicine. When nucleic acid is 2 chains, nucleotide change under arrangement turns into base pair change of arrangement. The term an "arrangement specific oligonucleotide" points out an oligonucleotide which carries out hybrid formation to specific arrangement which is not included or included in an allele, and these arrangement is specific to arrangement variation detected including base pair change detected. Depending on arrangement analyzed, one or more sorts of arrangement specific nucleotides can also be used for below still like a statement to arrangement of That . A term called [restriction fragmentation length many forms J (RFLP) where it is used for this specification points out a difference between individuals of the length of restriction fragmentation formed by digesting by specific restriction and nuclease. The term "thermostable enzyme" used for this specification is stable with heat, has heat resistance, and means enzyme which carries out the catalyst (promotion) of the combination of a nucleotide by a suitable method of forming a complementary primer extension output in each nucleic acid chain. Generally, composition is production To shake about a molecule of length which advances and is different in the direction of 5' along with a mold chain until it is started at the 3' end of each primer and composition ends. However, composition is started at the 5' end and there is also thermostable enzyme which advances in other directions using the same process as the above. The thermostable enzyme used for this specification must fulfill a standard to Only of being effective for an amplification reaction, and when time high temperature required for enzyme to denature 2 chain nucleic acid is given, don't denature irreversibly (inactivation). Irreversible denaturation in this case means losing enzyme activity eternally and completely. Although heating conditions required for denaturation change, for example with length and nucleotide composition of salt concentration of buffer solution, and nucleic acid which denatures, typically, they are the range of abbreviation 90~about 105-degreeC, change mainly with temperature and the length of nucleic acid about time, and are for about 0.5~4 minutes typically. High temperature can also be used if salt concentration of buffer solution and/or GCMi Formation of nucleic acid increase. Preferably, enzyme does not denature irreversibly by about 90~100 degreeC. Although it is a temperature higher the optimal temperature as which thermostable enzyme used for this specification is preferred, and the enzyme functions than about 40 degreeC and lower than temperature by which a hybridization of a primer to a mold is promoted, (1) Depending on composition and length of magnesium, salt concentration, and (2) primers, the hybridization can happen at a still higher temperature (for example, 45~70degreeC). If the optimal temperature for enzyme becomes high, primer-related singularity and/or selectivity of the elongating method will become large. However, enzyme active at less than 40 degreeC (for example, 37degreeC) also comes [Is stable?] to heat, and is contained within the limits of * To and the present invention. Preferably, the optimal temperature is about 50~90degreeC, and is 60~80degreeC still more preferably. Thermostable enzyme used for this specification can also be obtained from what kind of the origin, and may be nature or recombination protein. An example of enzyme reported to literature as what has heat resistance, Polymerase (Thermus flavus) stable with heat, for example, Thermus flavus of thermophilic bacteria, Therms Thermophilus (Thermus Thermo hilus), Bacillus Stearothermophilus (Bacillus sLearothermohilus) (it has the optimal temperature a little lower than what has been reported to other literature), Thermes Aquaticus (Thermus Fertilization I Samurai -" ( Translocation ) and Thermes rough terrace (Thermus Iacteus) Thermes Rubens (Thermusrubens)) And there is polymerase extracted from methano Thermus Suhr-Bidus (Methanothermus fervidus). Desirable thermostable enzyme used for this specification is Thermes Aquaticus (it is the DNA polymerase isolated from Thera+us Muslim animal .). the stocks of various kinds of can also be obtained from American Type Culture Collection (American Type Cu1ture Co11ection) Rockville and Maryland -- 07 tee day The (T, D, Brock), J, and Bact. It is indicated to 117 volumes and 189~196 pages in 98 volumes, 289~297 pages, tee Oshima (T, Oshima), Arch, Microbiol, and (1978) in (1969). One of the desirable strains of these is YT-1 share. In order to collect natural protein, a cell is proliferated using suitable art. Such art is indicated to Cardin et al. (Kaledin), Red crimson 1L, and the same as the above (1980). If it says simply, it will be Nitorirolotri acetic acid (100Qr) to 1 liter, Lipton (ag) yeast extraction liquid (ag), succinic acid (5g), sodium sulfite (50fff), riboflavin (ITIN), and KtHPOn (522nv>, Mg5Oa (48011W), CaC1t (222ag), NaCj!) (20 mg) And a cell is proliferated on a culture medium containing an element of the amount of traces. pH of a culture medium is alike and it is adjusting it to 8.0±0.2 by OH. If it cultivates to 20g to 1 liter of cells, aerating violently at temperature of 70 degreeC, a yield will increase. Cells (it measures by absorption by 550nto) in a second-half logarithmic growth phase were collected by centrifugal separation, and it washed with buffer solution, and carried out freeze preservation at -20 degreeC. By another method of growing a cell to be depended on the same as the above in Cheno et al. (chien), J, Bacteriol, and 1976, A defined mineral salt culture medium containing 0 or 3% of glutamic acid which filled up biotin of 0.1ntr/7!, O, Img/l thiamin, and nicotinic acid of 0.05 mg/l is used. In these salt, they are Nitorirolotri acetic acid and Ca5Ot+Mg5Oa and Na (/!). It is in KNOx, NaN0z, Zn5Oa, HJOzCLISO4, NaMoO4, CoC1z, FeC1-r, and MnSO4 and NatHPo. pl of a culture medium is adjusted to 8.0 by NaOH. In a method of Cheno and others (chien), a cell is proliferated in a bathing shaker by 75 degreeC at first. If fixed concentration is reached, it will put into a hot wind incubator, will come out, and will move 1 liter of these cells to a certain 16-liter cowboy (carboys). It lets sterile air pass in culture solution, and maintains temperature to 75 degreeC. Centrifugal separation recovers, after proliferating a cell for 20 hours. Isolation and generation of enzyme are performed after multiplication of a cell in six steps, and each stage is performed by a temperature lower than room temperature, preferably about 4 degreeC. In a stage or a process to The , when a cell has frozen, it thaws, and crushes ultrasonically and pH is suspended to about 7.5 buffer solution. In the second phase, supernatant liquid is collected and it classifies by adding salt like dry ammonium sulfate subsequently. Suitable halving (typically 45~75% saturation) is collected, and it dissolves in 0.2 potassium-phosphate buffer solution, preferably pH 6 and 5 buffer solution, and dialyzes to the same buffer solution. In the third phase, nucleic acid and protein of a certain kind are removed. Halving from the second phase is applied to a DEAE-cellulose column balance-ized with the same buffer solution as the above. Subsequently, this column is washed with the same buffer solution, it is eluted, parts for protein content drawing are measured and collected by absorption by 28 Or+m, and it has the same ingredient as what set pH to 7.5 with buffer solution to The preferably to 10mM potassium phosphate buffer solution, and dialyzes. In the fourth phase, it applies to a hydroxy apatite column which made a balance Fractionation collected as mentioned above with buffer solution used for dialysis in the third phase. Subsequently, this column is washed and enzyme is eluted by a straight line gradient of O9 old M~0.5M potassium phosphate buffer solution of pH 7 and 5 containing 2-mercaptoethanol of 10mM, and 5% of glycerin. A part for drawing containing pooled thermostable enzyme (for example, DNA polymerase) activity is dialyzed with the same buffer solution as having used it for dialysis in the third phase. In the fifth phase, it applies to a DEAE-cellulose column which balance-ized a part for dialyzed drawing with buffer solution used for dialysis in the third phase. It is K (enzyme is eluted by a straight line gradient of buffer solution like f.) of 0.01~0.6M in buffer solution which washed this column next and was used for dialysis in the third phase. 100 minutes which carry out Activity of thermostable enzyme were examined about mixing of deoxyribonuclease (and and exonuclease) using a suitable method. For example, and after nuclease activity incubates with superfluous DNA polymerase, it can be measured by electrophoresis from change of a molecular weight of phage lambdaDNA or super coil plasmid DNA. Similarly, after processing exonuclease activity using a restriction enzyme which cleaves by some parts, it can be measured by electrophoresis from change of a molecular weight of DNA. Halving determined that it will not have deoxyribonuclease activity is pooled, and it dialyzes with the same buffer solution as having used in the third phase. In the sixth phase, a part for pooled drawing is put into a phospho cellulose column which has fixed Bodd boium . This column is washed and enzyme is eluted by pH 7 and 5 by a straight line gradient of buffer solution like KCl of 0.01in potassium phosphate buffer solution~0.4M. heat -- a part for pooled drawing which has stable polymerase activity and does not have deoxyribonuclease activity -- p. It dialyzes with buffer solution of 8.0. A molecular weight of dialyzed output can be determined by a method according to SO5PAGE, etc. using a protein molecular weight marker. Thermes Aquaticus which is one of the desirable enzyme (a molecular weight of DNA polymerase by which T h e r m u s 7 Calah refining was carried out is determined as about 86.000~90.000 Dalton by a described method.) A gene of thermostable enzyme of the present invention which codes this enzyme is Thermes Aquaticus (Thermus a3g.). When clone-ized from aticus genome DNA, it can also be made to produce by recombinant DNA technology. A perfect coding sequence about Thermes Aquaticus (Taq) polymerase, It can guide from bacteriophage C135:Taq# 4-2 on Bgl II -Asp718 (portion) restriction fragmentation of about 3.5 kb (s) contained in genome DNA insertion fragmentation of about 18 kb (s) (kilo base). This bacteriophage is deposited with American Type Culture Collection (ATCC) on May 29, 1987, and has deposition number No. 40.336. This gene is plasmid pFc83 (ATCC No. 67.422). Bgl II -Hind III restriction fragmentation of about 750 base pairs (bp) isolated from deposition on May 29, 1987, A residential area can be developed by connecting with about 2.8 kbHrnd II [-Asp718 restriction fragmentation isolated from plasmid pFC85 (ATCC No. 67.421, May 29, 1987 deposition). Although pFC83 restriction fragmentation has an amino terminal end of a Taq polymerase gene, restriction fragmentation from pFC85 has a carboxy terminus. Therefore, if such two fragmentation is connected with a vector which has suitable control arrangement and which was digested by corresponding, translation of full length Taq polymerase will be obtained. It was also found out that all the coding sequences of a Taq polymerase gene do not need a thing which have desired enzyme activity and for which active gene output is collected biologically. In polymerase analysis, it is production Did do which has about 1/3 [are the deficiency of an amino terminal end and absent] of a coding sequence about a completely active gene output. In addition to deficiency of N-end, each amino acid residue in a peptide chain which constitutes Taq polymerase can be changed by oxidization, reduction, or other derivative-ization, and cleaves this protein, and can obtain fragmentation holding activity. Change which does not be not destroying activity does not except DNA arrangement which codes the above-mentioned protein from a definition of a gene. Therefore, change of the primary structure by deficiency of amino acid included in arrangement between translations, addition, or change itself can be performed, without destroying proteinic activity. Change of this substitution or others brings about protein which has the amino acid sequence coded by DNA which exists within limits the present invention was expected to be. Polyclonal antiserum from a rabbit which carried out immunity by polymerase of 86, 000~90.000 Dalton by which the present invention was refined is used, and it is Thermes Aquaticus (the probe of the partial genome expression library of Thermus 7- was carried out, and a suitable coding sequence was acquired as follows.). It can be made directly revealed [using control arrangement of itself in making it revealed as fused polypeptide Or ], or a clone-ized genome sequence can be made to reveal by composition using suitable control arrangement for a specific host used for revelation of enzyme. Of course, DNA availability which codes these arrangement provides an opportunity to make it produce Mutin (mutein) type which changes codon arrangement and also has DNA polymerase activity. For example, these means can provide a perfect coding sequence for TaqDNA polymerase, and can carry out expression vector composition of what can be accepted by various kinds of host systems after this, and can reveal a coding sequence. It is clear from the above thing that a portion's of a Taq polymerase coding sequence it is useful as a probe and also useful as a probe for collecting heat stable polymerase coding sequences of others of various kinds. Therefore, they are E and Cori (E.) about a portion of genome DNA which codes at least six amino acid. It can reproduce in coli, And a thing of a form which changed used as a probe used in order to collect DNAs of others which code at least six amino acid, and code heat stable polymerase, or an oligo deoxyribonucleotide probe is compoundable. Since nuclease arrangement in Thermes Aquaticus (Thermus Mr. Jing Dynasty ) and arrangement of a portion to which other kinds correspond may not be correctly in agreement, (Six amino acid A strange are coded) Probably, oligomer which has about 18 nucleotides will be required to perform a hybridization under sufficient stringency conditions to eliminate a wrong positivity. Arrangement which codes six amino will supply sufficient information for this probe. What And in a general way of speaking, manufacture of rearranged type Taq polymerase includes the following processes. In the first place, it is mature enzyme (in this case, used so that all the Mutin may be included), Or DNA which codes a fusion object with arrangement of an addition which it can cleave under a fusion object of Taq polymerase and arrangement of an addition which does not destroy the activity, or controlled conditions (as [base / on processing with peptide / for example, ]), and can bring about other protein is obtained. When arrangement is not interrupted by the intron, it is suitable for revelation in any hosts. This arrangement should be a form possible [logging] and callable. Next, preferably, a coding sequence started or collected is connected so that suitable control arrangement and an operation in an expression vector which can be reproduced are possible. Using this vector, a suitable host is transformed, a transformed host is cultivated under desirable conditions, and recombination Taq polymerase is made to produce. Arbitrarily, although 'l'aq polymerase is isolated from culture solution or a cell, when a certain kind of impurities are permitted, recovery and refining of protein are not sometimes required. Each of the above-mentioned stage can be performed by various kinds of methods. For example, a desired coding sequence can be acquired from genome fragmentation, and it can also use for a suitable host directly. Development of an expression vector which can act by various kinds of hosts is performed using the suitable following replicons. When it cannot usually use, in addition to an end of a coding sequence, the suitable restriction site can obtain a gene which can be started, and can carry out Extract ON to these vectors. Control arrangement, an expression vector, and transformation procedure change with models of a host cell used for revealing a gene. Generally, a procaryotic cell, yeast, an insect, or a mammalian cell is useful as a host now. For production of recombination protein, an original core host is the most effective, convenient, and, so, generally preferred for revelation of Taq polymerase. Also in any under a recombination condition and natural conditions, in a specific case of Taq polymerase, deficiency happens considerably at the proteinic N-end, and proof which shows that proteinic activity is held in addition exists in it. Separation (ed) natural firefly white matter is a result of Degradation by protein decomposition, and is not a result of translation of an imperfect gene. Mutin produced from an imperfect gene of plasmid IFe12 is completely active like Mutin produced from DNA which codes arrangement which consists perfect length by analysis of DNA polymerase, carrying out a deer. Since active thing of a form where a certain kind of N end was shortened is clear, revelation of genetic constitution or polymerase used may also consist a coding sequence of shortened corresponding shape. ' Distribution I and Response Most generally, a To * procaryote is represented by various stocks of E and Cori (L Auspiciousness ). However, other culture of the microorganism like bacillus Genus (Bacilus 5ubtilis), for example, bacillus Subtilis, various kinds of Sud monas (Pseudomonas), or other strains can also be used. In this original core system, a host, a replication site originating in a kind of conformity, and a plasmid vector that has control arrangement are used. For example, typically, E and Cori (Lcoli) will be transformed in poly bars (Bolivar), Gene, and 1977 using a derivative of pBR322 which is a plasmid originating in two volumes, E by 95 pages, and (Lcoli) the Cori kind. pBR322 has a gene of Ampicillin and tetracycline resistance, and when it develops a desired vector, it provides an additional marker held or destroyed. In original core control arrangement for which this specification statement which promotes a transfer start, and has an operator arbitrarily, and has ribosome binding site arrangement is generally used, beta-lactamase ( Benicillinase ), and lactose (I a c) promotor system [Chang (chang) et al., 198 (1977) Nature (s), 1056page], tryptophan (trp) promotor system [Godel et al. (Goeddel), Nucleic Ac1ds Res, eight (1980) volumes, 4057 pages], PL promotor [ Shimatake et al. [and] of lambda origin (Sh ima nucleic acid ke), There is an N-gene ribosome binding site useful as a control cassette in which 292 (1981) Nature (s), 128 pages], and transportation are possible, It is made useful as a porter pull control cassette, and this is this thing, the [which is PL promotor who connected with the second DNA arrangement corresponding to the NI?BS upper stream of the third DNA arrangement that has at least one restriction site which enables cleavage within N□ and 6bp3' of arrangement operational] -DNA arrangement is included. Phospha Tarta A which was written in the European patent laying-open-of-application No. 196, 864 specification (October 8, 1986 issue) of Chang (chang) and others, and was inherited by the same successor (phoA) a system is also useful. However, a promotor in whom what kind of use of conformity is possible can also use for a procaryote. In addition to bacteria, a eucaryotic microorganism like yeast can also be used as a host. Although most baker's yeast which is a laboratory strain of Saccharomyces cerevisiae (Saccharom ces cerevisiae) is used, many of other strains can also generally be used. It is [brooch 1 although a To have vector for 2-micron initiation sites of replication is shown. Jay, R (Broach.) other suitable plasmid vectors for revelation of J, R, Meth, Enz, 101 (1983) volumes, 307 pages], and yeast are known -- [-- for example Stink sea weeds (Stincbcoab) and 282 (1979) Nature (s), 39 pages, Chambe et al. (Tschea+pe), ten (1980) Gene (s), 157 pages, Clark et al. (c1arke), Meth, Enz, 101 (1983) volumes, and 300 pages should be referred to.] L Hess et al. (Hess) in whom control arrangement to a yeast vector has a promotor of composition of glycolysis enzyme, Mur ratio MP 5" top " (1968) 7@, 149 pages, Holland (Holland) et al., and 1 Fallen 14 -- 1y Castle l 17 (1978) volume and 4900page]. For a promotor known by these other industries, 3-phosphine Grise rate kinase ( Hivseman et al. (Hitzeman)) J, Biol, Chem, 255 (1980) volumes, 2073 pages, and other glycolysis enzyme, For example, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, Ruby decarboxylase and phosphofructokinase, glucose 6-phosphate isomerase, 3-phosphoglycerate mutase, pill Beat . There is a promotor of kinase, Trioce phosphate isomerase, phospho glucose isomerase, and glucokinase. Other promotors who have an advantage of an addition controlled by multiplication conditions, He is a promotor of enzyme about a dialytic ferment related to alcoholic dehydrogenase 2, iso Chitoku loam C, acid phosphatase, and nitrogen metabolism, malt sugar, and galactose use (Holland (Holland) same as the above].). Terminator arrangement is considered to be desirable in 3' end of a coding sequence. This terminator is found out by 3' untranslation region following a coding sequence in a gene of yeast origin. Enolase gene [Holland et al. (Holland, M, J) and J in which many of shown vectors have plasmid peno46, Biol. Although it has the control arrangement guided from LEU 2 gene [brooches (Broach, J), eight (1978) buttons ne, and 121 pages] obtained from Chem, 256 (1981) volumes, 1385page], or YEp13, Any vectors which have a yeast conformity promotor, an initiation site of replication, and the System ?'fil arrangement of It was are preferred. Of course, it is possible to also make a gene which codes polypeptide in a true core host cell culture medium originating in a multicellular organism reveal. For example, please refer to Patternin (Pa t terson) editorial supervision (1973) with Ti5sueCulture % Academic Press (Academic Press) and Gurko (cruz). There are rat myeloma N51, VEI?O, helium1a$ [lT A cow , and a Poisonous rat ovary (cHO) cell in a useful host cell line. An early stage and a second-half promotor ( Fields et al. (Fiers)) by whom an expression vector of these cells is usually used for - boat fishing from simian virus 40 (SV40) 273 (1978) Nature (s), 113 pages, or a polyoma virus, It has a promotor and control arrangement of conformity in a mammalian cell like a promotor who originates in adenovirus 2, cow papilloma virus, or a Trizal coma virus or an immunoglobulin promotor, and a heat shocking promotor. A DNA revelation system in a mammals system using BPV as a vector is indicated by United States patent 4th and No. 419.446 specification. Modification of this system is written in a U. S. Pat. No. 4, 601.978 specification. A general mode of transformation of a mammalian cell system is written in a U. S. Pat. No. 4, 399.216 specification. Although an "enhancer" field is considered to be important in the optimal revelation, these fields are arrangement generally found out upstream of a promoter region. If it is a request, an initiation site of replication can be obtained from a virus source. However, inclusion for a chromosome is a general mechanism with a DNA duplicate in a eucaryotic organism. Control arrangement which can also use a plant cell as a host and suits a plant cell, for example, a Tubalin synthase promotor, the Pori Ade Nils-ized signal sequence ( Depicso car et al. (Depicker, A), J, Mol.) phantom" ground- Convergent 1 (1982) -- one volume and 561 pages can be used. [mirror it has been reported that a revelation system using an insect cell which uses a control system provided by Bamboo virus vector these days is also (Miller.) D, W et al., Genetic En 1neerin (1986), Setlow editorial supervisions (Setlow and J Into ), Blenheim publishing (Plenum Publishing), eight volumes, 277~297 pages]. These systems have succeeded also in production of Taq polymerase. Depending on a form 1 5 To use host cell, transformation is performed using suitable standard art for the cell. Calcium handling (Cohen (cohen, S, N), Proc, Natl, Acad, Sci (USA), 69 (1972) volumes, 2110 pages) using a calcium chloride is used for a cell which has a procaryote or other substantial cell barriers. agrobacterium Tumefasi -- infection [Siau [being nth (A robacterium tumefaciens) ] et al. (Shaw, C, H), 23 (1983) Gene (s), and 315 pages are used by a certain kind of plant cell. the [■ 2 area with Graham (Graham), Van Dell, and the calcium phosphate precipitating method of nib (van der Eb) preferred in a mammalian cell without the above cell walls -- ■ (1978) 52 volume and 546page]. Transformations to yeast are Vanzolingen et al. (VanSolingen, P) (J, Bact, 1977, 130 volumes, 946 pages) and Zhao (Hsiao.). It is carried out by a method of C, L and others (Proc, Natl, Acad, Sci, (USA), 1979, 76 volumes, 3829 pages). A method of isolating in DNA which codes protein of a lambda tl - Eve - ■-0'' request, for example, DNA which codes Taq polymerase, using bacteriophage lambdagtll is as follows. A library produces by perfect digestion of Thermes Aquaticus (Thermus a uaticus) DNA, [young (Young) and Davis (Davis) who have at least one copy of EcoR inserted in an EcoRI part of lambdagtll phage to both ends and who may comprise Alul fragmentation, Proc, Natl, Acad, Sci, USA s1983, 80 volumes, 1194~1198i. DNA inserted since at least one copy of unique EcoR in this bacteriophage arranged to a carboxy terminus of beta-galactosidase gene (in a suitable frame and a direction), It is revealed as protein united with beta-galactosidase under control of a lactose operon promotor / operator. Next the antibody plaque hybridization method is used and a genome expression library is screened. This method is called "epitope selection" and identifies high A bitch and a plaque which carried out Do formation using an antiserum to fused protein arrangement coded by this phage. For example, a library of this recombination phage can be screened using an antibody which recognizes Taq polymerase of 86, 000~90, 000 Dalton, and phage which consists a DNA segment which codes an antigen determinant of this protein can be identified. Recombination phage is screened using all the rabbit Taq polymerase antiserums for about 2X10 s. In this - next screening, it is refined from a candidate plaque which a positive signal is detected, and one or more sorts of these plaques do not react to preimmune serum, and reacts to an immune serum, and is analyzed a little in detail. In order to inspect fused protein produced by recombination phage, a lysogenic strain of phage in host Y 1089 is obtained. A lysogenic strain is induced, after carrying out gel electrophoresis of the protein to generate, that of production To shake can be observed or duplication arrangement may produce the new protein in which each lysogenic strain is not looked at by other lysogenic strains. A factor which has a positive signal is taken out. A positive plaque was taken out in an example indicated here, it further identified, the re-plate was carried out with low density, a Recombination object was purified, and a size class by digestion by an EcoRI restriction enzyme analyzed a purified clone. Next, a probe is made from a DNA insertion sequence which isolated, a sign is performed suitably, and they are Maniatis (Maniatis) et al. and Mo1ecular Cloning about these probes : A Laboratory Manual. It can use for usual plaque no Idealization or a colony hybridization analysis method indicated in 1982. The probe of the second genomic library constituted in a Sharon (charon) 35 bacteriophage was carried out using a probe which carried out the sign (oui, Ruhr mines (Wilhelmine, A, M), Gene, 1983, 26 volumes, 171~179 pages). this library -- Thermes Aquaticus (fragmentation (15~20kb) which was made by S au3A partial digestion of Therlous Dandy Yutani genome DNA, and carried out size judgment -- Sharon 35 -- it clone-ized to the furzy Ban old part.) Phage containing DNA which codes Taq polymerase was isolated using this probe. CH35: It became clear that one of the phage which named it Taq#4-2 and to generate had all the gene sequences. Partial arrangement which codes a portion of this gene also isolated. Development of a suitable vector containing a coding sequence and control arrangement of a In addition, 2 Inquiries request uses well-known standard connection and restriction art in this industry. It cleaves, a plasmid which isolated, and an oligonucleotide which was DNA-arranged or was compounded are prepared and re-connected, and it is made desired shape. Site-specific DNA cleavage is performed by processing using a suitable restriction enzyme (one or more sorts) under well-known conditions in this industry, and the details are indicated by manufacturer of a restriction enzyme of these marketing. In for example, 2 Knee England bio-lapse (New England Biolabs) and - boat fishing by which a product brochure should be referred to, He cleaves a plasmid or DNA arrangement of about 1 river with enzyme of one unit in buffer solution of about 20 tll (s), and is trying to digest a DNA substrate completely typically in an example of this specification using an excessive quantity of restriction enzymes. It can also change, although about 1~2 incubation hours are effective at about 37 degreeC. After each incubation, phenol/chloroform can extract protein, it can remove by subsequently performing ether extraction, and ethanol precipitation can recover nucleic acid from water part drawing. If it is a request, polyacrylamide gel or agarose gel electrophoresis using standard art can perform size separation of cleavage fragmentation. General explanation of size separation is indicated to 65 volumes and 499~560 pages in MethodsinEnzymology and 1980. About fragmentation which carried out restriction cleavage, it is in tris of 50mM, NaCn of pH 7, 6, and 50 mM, MgC'z of 10mM, DTT of 10+1M, and dNTPs of 50~Loom, Incubation time for about 15~25 minutes is used by 20~25 degreeC, It can be made a flush end by processing in existence of four kinds of deoxy nucleotide triphosphate (dNTP) by E and large fragmentation of the Cori (E, coli) D N A polymerase I (Freneau (Klenow)). Although the Freneau fragmentation carries out the fill-in of the 5' adhesion end, even if four sorts of dNTPs (es) exist, it acts as Chu Buck (chewsback) of the 3' single chain to project. If it is a request, selective repair can be performed by supplying only I sorts or a plurality of selected dNTPs (es) within restriction specified by quality of an adhesion end. After processing by Freneau, phenol/chloroform extracted a mixture and it made it precipitate by ethanol. If it processes using 31 nucleases under suitable conditions, hydrolysis of a single chain portion will take place. An synthetic oligonucleotide can be prepared using an automatic synthetic method, using a triester method of Mateuachi and others (Matte-ucci) (J, Am, Chem, Soc, 1981, 103 volumes, 3185~3191 pages). Kinase processing of a single chain for a sign before annealing, It carries out using an excessive quantity, for example, polynucleotide kinase of about ten units, to a substrate of 1nM under existence of tris of 50II+1, MgCj!z of pH 7, 6, and 10 mM, dithiothreitol of 5mM, and ATP of 1~2mM. When this kinase processing is for a probe carrying out a sign, ATP has T 3Zp of high-pressure activity. A capacity of 15~30 m performs connection at following standard conditions and temperature. 20mMTris-HCj!Na (J) of 10 (pH 7, 5) mM MgC1t and 10mM DTT133i/ a / B5A110mM~50mMAnd (connection of an "adhesion end") ATP of 40 Oh and 0.01~0.02 T4DNA ligase of a unit (Weiss) (Weiss), 0 *C; or (connection Niha of a "flush end") 1mM (7) ATP, and 0.3~0.6 T4 [INA ligase of a unit ('Weiss (Weiss)), 14degreeC. Connection in a molecule "adhesion end" is usually performed at the total DNA concentration (the total end concentration of 5~1100 n) of 33~100 codfish / m/. Flush end connection between molecules (superfluous Mol's 10~30 times as many linkers are usually used) is performed at the total end concentration of 1 Boiled . In development of a vector using "vector fragmentation", vector fragmentation is usually processed by bacterial alkaline phosphatase (BAP), 5' phosphate is removed, and re-connection of a vector is prevented. BAP digestion is pH 8, and in tris of about 150 mM (s), it is existence of Na" and Mg-2 and it performs it by 60 degreeC for about 1 hour using BAP of about one unit of vector 1■ hits. In order to collect nucleic acid fragmentation, phenol/chloroform extracts manufacture things and it carries out ethanol precipitation. By a vector in which double digestion was carried out by additional restriction enzyme digestion, the connection can prevent fragmentation which is not preferred. Yu Lord "" Draw -- Corp. -- about a portion of a vector originating in cDNA or genome DNA which needs change of strange arrangement again, site-specific primer instruction variation indecement is used. This art is standard in this industry now, and is performed to single chain phage DNA by which variation indecement should be carried out except for a limited mismatch showing a desired variation using a complementary primer synthesis oligonucleotide. If it summarizes, 2 chain DNA to generate will be transformed into host bacteria of phage bearing properties, using an synthetic oligonucleotide as a primer which orders phage composition of a complementary chain. A plaque is made to form from a single cell which applies culture solution of transformed bacteria to upper N agar, and has phage. Theoretically, it has the arrangement of a basis 50% including phage in which 50% of new plaques have a variant as a single chain. It is the temperature which is enough to move a plaque to a nitroglycerine cellulose filter, and make possible a hybridization which Matches correctly, and for a mismatch with a chain of a basis prevent a hybridization, It is [lift (an IiftsN hybridization is performed.) at a synthetic primer which carried out kinase processing. Next, a plaque which carries out high Fried formation with this probe is extracted and cultivated, and DNAs are collected. In development of 1 or less Prostitution , a connection mixture is first used for exact connection of a plasmid development thing, and they are E and Cori (E.). It confirms by transforming 294 shares of co I i MM (s), or other suitable hosts. It depends for a transformant of a good result on a method of plasmid development, and is Ampicillin tolerance as understood in this industry, He is Frwell et al. (clewell, D, B) (Proc, Natl.) about 6 chosen by tetracycline resistance or other resistance to antibiotics, next a plasmid from a transformant, Depending on the case, it will prepare 62 volumes and a 1159-page method in Acad, Sci, USA, and 1969 by the chloramphenicol amplifying method ( Frwell (c1enel l+D, B), J, Bacteriol, 1972, 110 volumes, 667 pages). Restriction processing analyzes DNA which isolated and they are/, or Sanger et al. (Sanger, F) (Proc, Natl, Acad.). It is Sci, USA, 1977, 74 volumes, and a 5463-page dideoxy procedure, and he is Mehsing et al. (Messing) (NucleicAcids Res) further. In 1981, sequence determination will be carried out by nine volumes, method indicated by 309 pages, or a method of Maxam and others (Maxall) (Methodsin Enz 5olo, 1980, 65 volumes, 499 pages). Borrower -- the host strain used for cloning and revelation in this invention is as follows. E, E obtained from (E, coli) Cori Genetisoku stock center GC5G#6135, and the Cori interval between roots (E, coli) 294 were used for revelation of a development thing under cloning, sequence determination, and almost all bacterial promotor's control as a host. E, a Cori ( A time -coli) K 12-share MC1000lambda lysogenic strain, NJstC1857SusPgo, and ATCC39531 can be used for revelation under PLNlll+3 promotor's control. E deposited by ATCC (ATCC53606) in this specification on April 7, 1987. Cori DG (L juxtaposition) 116 is used. E and Cori (E, colt) it is easy to receive phage infection for an M13 phage Recombination object, for example, E. 12-share DG9B is used for Cori. ATCC deposition is carried out on July 13, 1984, and 98 shares of DG (s) have deposition number 39768. CO5-7, CO5-A2, CV-1, and rat bacteria can perform mammals Teno revelation, and it is based on revelation by the amount of Spodobutera full Dipeta °C group o d Together ra-red A rainbow in an insect cell. M1 Tokyo Lm Probation . Enzyme must be stored into buffer solution containing one sort or two or more sorts of nonionic polymer detergent, in order to use stability for a long period of time. Generally a range of a molecular weight is about 100~250, 000, preferably it is about 4, 000~200.000 Dalton, and, as for such detergent, pH stabilizes enzyme by abbreviation 3.5~about 9.5, preferably about 4~8.5. As an example of such detergent, it is Emulsifiers & Deter ents of hook Kuceon (McCutcheon), The North America version (1983) mc publishing company (MCPublishing Co) There are hook Kuceon section issue, 175, a lock load, a Glen lock, and a thing indicated to 295~298 pages of New Jersey (U. S.) , , and please refer to the above-mentioned literature for the details. Fatty alcohol ether and lauryl ether in which detergent ethoxylated preferably, Ethoxylated Alkyl phenol, an octyl Phenoxypoly ethoxy ethanol compound, It is chosen from a group which consists of straight-chain-shape alcohol, a polyethylene-glycols monooleate compound, a polysorbate compound, and phenolic fatty alcohol ether which were formed into Oxy propyl and/or it carried out property modification Oxy ethylation. Tween (Tween) 20 which is polyoxy ethylation (20) sorbitan monolaurate preferably more particularly (ICr and the Americus Yingkou Volatile (r (JAmericas Inc))) It is Iconol (Tconol) (registered trademark) NP-40 ( A bush (BASF) a Ian dot corporation, a Persistence bunny, new chassis) which is Wilmington, DE, and ethoxylation Alkyl phenol ( Nonyl ). Thermostable enzyme of the present invention is used for the object which this enzyme is required or is desirable. This enzyme is used for the following amplification protocols in specific desirable Mr. B. An amplification protocol using enzyme of the 1 finger 1 A time top uni-fire present invention is the method of amplifying the existing nucleic acid arrangement by which is indicated by the European patent laying-open-of-application No. 200, 362 specification, and the application for patent is carried out to it. However, as for this enzyme, using at the following amplification processes is preferred. - being related with the number of reaction stages of an amplification process being a chain reaction and participating in boat fishing -- logarithm -- at least one sort of specific nucleic acid arrangement is consisted of a production To shake chain reaction in quantity [-like]. however, (a) -- an end of arrangement [ Desire ] is known in detail enough, can compound to them an oligonucleotide which carries out hybrid formation, and on condition that arrangement of (b) small quantity can be used for starting a chain reaction, it carries out. Probably, output of a chain reaction is separate nucleic acid duplex which has an end corresponding to an end of a specific primer used. or [that it was refined or any nucleic acid arrangement of an unrefined form includes specific nucleic acid arrangement of it request] -- or when it is thought that it contains, it can use as start nucleic acid. Therefore, RNA which includes DNA or messenger RNA, for example can be used for this process, and these DNAs or RNA may be a 1 chain or 2 chains. What is a DNA-RNA hybrid and has each one chain of every can also be used. Nucleic acid produced from an amplification reaction preceded using a primer which can also use one mixture of these nucleic acid, or is the same or different can be used similarly. Specific nucleic acid arrangement which should be amplified may be a mere portion of a big molecule, or it exists as a separate molecule from the beginning, and the specific arrangement may constitute all the nucleic acid. Small-quantity part drawing of a mixture that arrangement amplified does not need to exist in a pure form, and complicated, For example, some beta-grotesque bottles (Psyche et al. (Saiki)) contained in all the human DNAsIt can also be a part of nucleic acid arrangement of a thing which is illustrated in 5cience, 230 volumes, 5-30~1534 pages of Work , and 1985], or a specific microorganism which constitutes only the minimum quantity portion of a specific biological sample. start nucleic acid arrangement has one sort or the specific nucleic acid arrangement of a plurality of requests, and this arrangement may be the same or may differ -- so -- this amplification process, It is useful also although one or more sorts arranged at a nucleic acid molecule which being not only useful but is so much the same as that of production To shake , or different in a certain kind of specific nucleic acid arrangement of different specific nucleic acid arrangement are amplified simultaneously. It can obtain from any origins, for example, one sort or two or more sorts of nucleic acid is pB (s) !? A plasmid like 322, nature of all the origins of DNA, RNA or bacteria clone-ized, yeast, a virus, an organelle and a plant, or higher forms of life like an animal -- it can also obtain from DNA or RNA. DNA or RNA can be extracted from blood, organization material, for example, chorionic villus, or an amnion cell by various techniques like a method indicated by Maniatis (Maniatis) et al., the same as the above, and 280~281 pages. When using a specific probe for arrangement which is amplified and is detected after that, A cell is generally heated to about 90~100 degreeduring 1~15 minutes C until it can use immediately, without extracting nucleic acid, they are suspended to a hypotonic buffer in this case and the dissolution of a cell and distribution of an ingredient in a molecule take place. An amplification reagent can be directly added to a cell which dissolved after a heating process. what kind of specific nucleic acid arrangement -- an amplification process -- production To shake -- things are made. A required thing is known in detail [a sufficient number of both ends of arrangement of bases] enough, Two oligonucleotide primers which carry out hybrid formation in a relative position which met arrangement with a chain with which desired arrangement differs can be prepared, When extension output compounded from one primer dissociates from the mold (phase assistant object), it is that while I will accept it to nucleic acid arrangement of demarcated length enables it to work as a mold for extension. Singularity of a primer for target nucleic acid arrangement can be made high, and efficiency of a process also becomes high as knowledge about a base of both ends of arrangement increases. The term a "primer" used after this can mean many primers from one sort, especially when information about 1 or end arrangement of a plurality of fragmentation amplified is a little indefinite. For example, a set of a primer which arrangement representing the diversity of all the possible codons by Degeneracy of a gene code has will be used about each chain, when nucleic acid arrangement is presumed from protein-sequences information. Probably, one primer which exists from this set will be as homologous as an end of arrangement of a request which should be amplified. An oligonucleotide primer can be prepared no matter suitable method [the above-mentioned phospho triester and the phosphodiester method, or / like those automated modes / what] it may use, for example. In such an automated mode, it may compound by a method (Tetrahedron Letters, 1981, 22 volumes, 1859~1862 pages) of view cages (Bea-ucage), using Diethyl phosphoramidite as a starting material. A method of compounding an oligonucleotide on an embellished solid base material is written in a U. S. Pat. No. 4, 458, 066 specification. It is also possible to use a primer isolated from the biological origin (for example, restriction and a nuclease digestive thing). Specific nucleic acid arrangement is produced, using nucleic acid containing this arrangement as a mold. About each different nucleic acid arrangement which a stage to The should be amplified in each nucleic acid chain, or should be detected, When nucleic acid which should be amplified or detected, including making four sorts of different nucleotide triphosphate and one sort of oligonucleotide primers contact, is DNA, nucleotide triphosphate is dATP, dcTP, dGTP, and TTP. A nucleic acid chain is used as a mold for composition of an additional nucleic acid chain. This composition can also be performed using what kind of suitable method. Generally, it is preferred, and pH is 7~9 and happens in water buffer solution which is about 8 most preferably. It is superfluous molar quantity (clone-ized nucleic acid) about two sorts of oligonucleotide primers to buffer solution which contains the separated molds preferably. usually -- it is a primer 2 mold ratio of about 1000:1 -- genome nucleic acid -- usually -- about -- it is a primer 2 mold ratio of to':t -- it adds. However, when using this method for a diagnostic use, quantity of a complementary chain may not be known, therefore it is understood that quantity of a primer to quantity of a complementary chain may be unable to be decided. However, in practice, generally, quantity of a primer added will turn into superfluous molar quantity to quantity of a complementary chain (mold), when arrangement amplified is included in a mixture of a complicated long-chain nucleic acid chain. In order to improve efficiency of a process, a large excessive quantity is preferred. Concentration of nucleotide triphosphate is 150~200 team in buffer solution for amplification, respectively, and MgCI-t makes efficiency and singularity of a reaction increase by existing in quantity of 165~2mM in buffer solution. Next, nucleic acid which should be amplified or detected processes solution to generate depending on 2 chains or a 1 chain. When nucleic acid is 1 chain, it is not necessary to use a denaturation stage and a reaction mixture is held at temperature which promotes a hybridization to the complementary target (mold) arrangement of a primer. Such a temperature is generally more than about 35degreeC~65degreeC or it, and preferably it is about 37~60degreeC, and effective time is generally for 0.5~5 minutes, and preferably is for 1~3 minutes. The singularity of a hybridization of a primer is made to increase preferably using 45~58 degreeC about a primer more than Taq polymerase and 15-Maher. Low temperature is more nearly required when a primer is short. A phase assistant object over nucleic acid of a chain is compoundable by adding 1 or two oligonucleotide primers to it in the - end of a basis. When a suitable single primer is added, primer extension output is compounded in existence of the primer, thermostable enzyme, and nucleotide triphosphate. This output is partially complementary to nucleic acid of the end chain of -, and generates a complementary chain with which hybridized with the nucleic acid chain, and formed duplex of a chain of length which is not equal, this was separated as mentioned above subsequently to a 1 chain, and two single What were separated. It may react by adding two sorts of suitable primers to 1 chain nucleic acid. When nucleic acid has two chains, before using this as a mold, it is necessary to separate a chain of nucleic acid. This chain separation can be performed by the suitable denaturing method like a physical, chemical, or enzymatic means. A desirable physical method which separates a chain of nucleic acid includes heating until nucleic acid denatures completely (99% or more). Temperature of typical heat denaturation is about 90~105'C, and time is generally for about 0.5~5 minutes. Preferably, an effective denaturation temperature is 90~100degreeC, and is for 0.5~3 minutes. Separation of a chain can also be induced with enzyme from a class of enzyme known as helicase, or enzyme RecA in which carrying out owner Perilla frutescens (L.) Britton var. crispa (Thunb.) Decne. of the helicase activity, and denaturing DNA under existence of Ribo ATP is known. Suitable reaction conditions to separate a chain of nucleic acid using helicase, Turn Hoffmann Bering (Kuhn Hoffmann-Berling), It is indicated in alpha-43 C3 H-Quanti nucleic acid tivel Fallen 1 phantoms, 63 pages, and 1978, and technique using RecA is indicated in See Rushing (c, Radding) Ann, Rev, Genetics, 16 volumes, 405~37 pages, and 1982. or [that it is equal by these denaturing methods] -- or two separated complementary chains of length which is not equal arise. When 2 chain nucleic acid denatures with heat, it cools radiationally to temperature which promotes a hybridization to complementary target (mold) arrangement of each primer which exists a reaction mixture. This temperature changes with reagents, it is usually more than about 35degreeC~65degreeC or this, and preferably is 37~60degreeC, and is generally preferably maintained for 1~3 minutes for 0.5~5 minutes effective time. In fact, about Taq polymerase, temperature is only preferably reduced to a 37 degreeC grade from about 95degreeC to about 45~58 degreeC, and a hybridization occurs at temperature within the limits of this. Even if nucleic acid is any of a 1 chain or 2 chains, thermostable enzyme can be added in a denaturation stage, or when temperature falls to a range which promotes a hybridization, or when it is in temperature of this range, it can add -- ranking next zeroIt heats to sufficient temperature for making the activity of enzyme increase to promoting composition of temperature by which the activity of enzyme is promoted or optimized in a reaction mixture, i. e., a hybridized primer, and primer extension output from a mold. Although this temperature must be enough to actually compound extension output of each complementary primer to each nucleic acid mold, Don't be high temperature, so that each extension output from that complementary mold is denatured (that is, this temperature is generally below about 80 degrees C~90 degreeC). A typical temperature effective for this synthetic reaction changes with types of enzyme mainly used and one sort, or two or more sorts of nucleic acid, and is generally about 40~80degreeC, preferably 50~75degreeC. Still more preferably, this temperature is about 65~75degreeC, when using DNA polymerase from Thermes Aquaticus (Thermusaaua t 1cus). Time which this composition takes changes mainly by complexity of temperature, the length of nucleic acid, enzyme, and a nucleic acid mixture, and is in for about 0.5~40 minutes or more than this, preferably a range for 1~3 minutes. If nucleic acid is long, generally a long time is needed more. Since it is found out that dimethyl sulfoxide (DMSO) checks the activity of Taq polymerase enzyme, DMSO does not need to exist or is not recommended. A newly compounded chain and its complementary nucleic acid chain form a molecule of 2 chains, and this molecule is used in a stage where this process continues. In the following stage, a chain of 2 chain molecules is separated by carrying out heat denaturation at temperature which is not so high as thermostable enzyme denatures completely irreversibly or inactivates 2 chain molecules although it is effective for denaturing however. This temperature changes mainly with a type of enzyme, and length of nucleic acid, and in -Jn, about 90~105degreeC and time which is 90~100degreeC still more preferably and denaturation takes change mainly with temperature and the length of nucleic acid, and is for 0.5~4 minutes typically. Temperature is lowered to a primer after this processing time to a level which promotes a hybridization of a primer to 1 chain molecule (mold) generated from a stage of a complementary front. This temperature is the temperature which was described above. It adjusts after this hybridization stage or to temperature which can promote the activity of thermostable enzyme and can compound primer extension output instead of a hybridization stage (or simultaneous with it) using as a mold a chain newly compounded from a front stage. This temperature must not be as high as it separates extension output from that mold as described above (denaturation), either (it is usually for [1~] 3 minutes in for [0.5~4 minutes], preferably 50~70"C at 40~80 degreeC). Since a hybridization happens into this stage, when performing simultaneously such two or more stages of not being required for after-denaturation cooling of a front stage, a desirable temperature range is 50~70'C. As for heating and a cooling stage of composition of separation of a chain, a hybridization, and extension output, only the number of times required for that of production To shake can repeat specific nucleic acid arrangement of the amount of requests, and this number of times changes by a final use. This number of times is only restricted by quantity of an existing primer, thermostable enzyme, and nucleotide triphosphate. As for these stages, repeating twice at least is preferred. In order to use it for detection, when 0 which changes, for example according to quality of a sample, for example, a sample which should be amplified, is pure as for the number of cycles, there are few cycles and it ends. When a sample is a complicated mixture of nucleic acid, in order to fully amplify a signal and to detect it, more numbers of cycles are needed. To general amplification and detection, it is preferred to repeat a process at least 20 times. When using as follows an arrangement specific probe which carried out the sign, it is preferred to repeat these stages at least 5 times. When using human genome DNA with the above probes, this process is repeated 15~30 times preferably, arrangement is fully amplified, and it is made for a detectable signal to generate clearly. That is, a bank ground noise is kept from barring detection. Quantity of generated specific nucleic acid arrangement is exponentially accumulated so that it may explain still in detail below. Unless enzyme does not denature or inactivates irreversibly, after adding a nucleotide, a primer, or thermostable enzyme first, it is not necessary to further add. When enzyme receives irreversible denaturation or inactivation, it is necessary to fill up enzyme after each denaturation stage. However, even if it adds these substances in each stage, it does not have a bad influence on a reaction. Making many specific nucleic acid arrangement produce from one sort from a mixture of nucleic acid to The or nucleic acid uses an oligonucleotide primer of a kind from which a number suitable occasionally which is a request differs. For example, when making two kinds of different specific nucleic acid arrangement produce, four kinds of primers are used. It is specific to one of the nucleic acid arrangement with specific two of these primers, and two sorts of remaining primers are specific in the second specific nucleic acid arrangement. In this case, each of two kinds of different specific arrangement is exponentially produced by the present invention. After time of suitable length passes and specific nucleic acid arrangement of the amount of requests is produced, a reaction is stopped inactivating enzyme by a known method (for example, addition of EDTA, phenol, SDS, or CHCN 1), or by separating an ingredient of a reaction. An amplification process may be performed continuously. In one mode of the automating method, a temperature cycle set up to control temperature for a predetermined period to the predetermined level may be performed to a reaction mixture. One device for this object is a device with which it automated for controlling an amplification reaction of the present invention. This device uses a fluid handling system controlled by a computer, and makes the second container in which temperature is controlled by a computer and that is in agreement with a prescribed incubation method carry out fluid movement of the enzyme kept at controlled temperature which changes with a container to The . This second container has stored nucleic acid arrangement, nucleotide triphosphate, and a primer of one sort or plurality which are amplified. A computer is provided with a user interface and can input now a process parameter in which a user controls the feature of various stages in amplification processes, such as time of an incubation, temperature, and quantity of enzyme to which it is made to move, by it. Since it is not necessary to move enzyme for every cycle, a desirable device which can be used uses temperature circulation without a fluid control system. Such a device consists of the following systems. A thermally conductive container which are a test tube of 1 and a predetermined number, preferably a 500micro A test tube, and fixes a test tube containing a reaction mixture of nucleotide triphosphate, a primer, nucleic acid arrangement, and enzyme. It is a device which heats 2 and a thermally conductive container, and is cooled, and is held to a temperature higher than room temperature and low, This device heats a container, it has an input part which receives a control signal which controls temperature cooled or kept -- [-- such a device -- a Materials electronics products corporation (FIaterialsElectronics Products Corporation),] 3 the Belle Deere (Pel tier) heat pump or a hydrothermal switchboard put on the market from Trenton and a new chassis may be sufficient as, computer paraphernalia linked to an input part of an above deviceA device which is (for example, a microphone Lopro sensor controller), and generates a signal which controls automatically an amplification process, a temperature level and a temperature gradient, and timing. In another mode, enzyme used for composition of primer extension output is fixable in a column. Inside of this column and a heating coil can be continuously circulated for other reaction ingredients with a pump. Therefore, generated nucleic acid can be denatured repeatedly, without inactivating enzyme. Next, an amplification protocol is shown typically. Here complementary tN (chain D N A is used as nucleic acid in the end containing arrangement (S) of a request containing S"] and [S-] of two.) the 1st -- and extension of each oligonucleotide primer on a mold of a basis generates a DNA molecular product with new infinite length which stops only by one of the primers between each reaction cycle following this. Such output to be called "long life Acid '' from now on will be accumulated linearly. That is, probably, quantity which exists after a cycle of a certain number is proportional to the number of cycles. In this way, generated long life Acid will function as a mold of an oligonucleotide primer of one side between subsequent cycles, or another side, and will generate desired arrangement (So) or a molecule of [S-]. A chain reaction which these molecules will also function as a mold of an oligonucleotide primer of one side or another side, and will generate [S *] and [S-] further, and will so bring accumulation of [5] at an exponential speed to the number of cycles may be continued. A by-product generated by oligonucleotide hybridizations other than an oligonucleotide hybridization meant is not autocatalytic, and, so, it accumulates it at linear speed. It turns out that that a complementary strand of each [which is stopped by an oligonucleotide array of a primer of one space below] and another side generates is a specific nucleic acid chain (S) for which it asks. A stage of this process can be repeated infinitely and limited by only primers 1 and 2, an inducer, and existing nucleotide. Since a nucleotide of a basis is not reproduced, the quantity is uniformly maintained through all the processes. Since long life Acid is generated only from nucleic acid of a basis, the quantity increases linearly. Quantity of specific arrangement increases exponentially. That is, specific arrangement serves as a dominant kind. This is shown in the next table. This table makes efficiency of each cycle 100%, and shows a relative amount of a kind which exists in n cycles. 2l2 [of 0~n cycles of following space '] of 2 13 Work 3 415 1 15 32.75220 1 20 1.048 and 555n 1 n When a chain nucleotide is used as (2 Ll-n-1) a mold in the end of one, one long life Acid per cycle generates. After amplification of at least 1 cycle, by adding a set of complementary primers to internal (there is nothing at end) arrangement of arrangement amplified, arrangement after amplification of the number of times of predetermined and in predetermined arrangement can be amplified, and singularity of a reaction can be enlarged. Such a primer may be added in what kind of stage, and will offer an amplified primer with that shorter of this Chi . Although it has an un-complementary end, longer fragmentation can be prepared by using in front in amplification and using a primer and a primer overlapped a little. Specific nucleic acid arrangement is clone-ized, and this vector can be used for zero-order [which can be inserted in a suitable expression vector], a suitable host living thing can be transformed into it, and it can make it produce gene output of arrangement with a standard method of recombinant DNA technique using this invention method. This amplification process can produce a mixture of nucleic acid from the original mold nucleic acid, target amplification output expected, and non-target output of various kinds of backgrounds. Amplified output may be a mixture, when it has a plurality of target sequences which can be set to a terrorism junction diploid genome to whether it is the original mold DNA, or when there is a family of a related gene. These primers are changed, and a mixture of DNA generated by amplification reaction is prompt, and it can assist loan-ization in a unique target. In such change, a restriction site is contained each of a primer, or during arrangement amplified and clone-ized. Preferably, it is the same or it is preferred to introduce a different restriction site into 5' end of a primer, and to make it produce a restriction site at the two ends of amplified output. If suitable enzyme cuts, amplified output will be easily inserted into a plasmid or a vial vector, and will be clone-ized. By this cloning, amplified analysis or revelation of output of each instead of a mixture is attained. When a primer has the restriction site introduced into it, the same restriction site can be used for both primers. However, if different restriction sites are used, output can be inserted into a vector in a specific direction, and a plurality of insertion and insertion which arises from amplification only based on one side of two primers will be controlled. At the time of cloning to a chain arrangement vector, specific directivity is useful in the end of -, when clone-ized output should be revealed when using -0 chain hybridization A probe or. One method of preparing a primer is choosing primer arrangement different very slightly from a target sequence. A beautiful woman by whom each of a primer should be arranged is screened about homogeny over a suitable restriction site for a desired vector. For example, target sequences rCAGTATCCGA and JPrimer arrangement from which only what has a BamHI part, and one base are different is correctly Matches (ed) at the 3' end, It is chosen so that it may have arrangement and a restriction site which were changed near [the] 5' end (for example, it is rCAGgATCCGA and J and a small letter expresses a target sequence and a thing which does not Matches ). A primer carries out hybrid formation with the original target sequence, and arrangement changed into this minimum does not bar the capability to start a polymerization. After an amplification cycle to The , a primer is copied, and serves as a target and it Matches it correctly with a new primer. Output is made to cleave by a suitable restriction enzyme after an amplification process, a desalination column or a molecular weight chromatography column is passed, or a film is passed, A digest product (one or more sorts) containing amplification arrangement which makes separate a restriction digestive thing from the prevention side of connection like nucleotide triphosphate and salt depending on the case, and should be clone-ized becomes as [be / it / bacteriophage M13], and is inserted in a low Jung vector by ligation. A cloning vector may have selectable markers in -J'lQ, and may have a promotor further depending on the case. next -- carrying out sequence determination of this by a publicly known technique, when a gene codes protein -- and/-- or it can be made revealedSequence determination of the gene can also be carried out by adding a suitable primer complementary into a portion of a request by which sequence determination should be carried out again into an amplification process. This primer will form extension output and a grade of amplification of having this extension output will provide arrangement information. One method that I accept it in order to prepare a primer, 0 which consists of taking out 3' end of a primer from a target sequence, and adding a desired restriction site to 5' end of a primer -- for exampleA HindI [I part can be added and arrangement rcgaagcttCAGTATCCGA and J (however, a small letter is as the above-mentioned definition) can be made. Although an added base does not contribute to a hybridization in a cycle to The of amplification, it is Matches (ed) in a cycle after this. Next, it cuts by one sort or two or more sorts of restriction enzymes, and output finally amplified is clone-ized as mentioned above, and is made to reveal. Genes amplified may be human beta-hemoglobin or a human HLAD mouth, DR or DP-alpha, and a beta gene, for example. In a method of another not much efficient thing not desirable [so] and being a cloning method which cannot say, and using flush end connection rather than adhesion (restriction enzyme is used) end connection, A fundamental amplification method is used regardless of a restriction enzyme in one sort which should be clone-ized, or two or more sorts of arrangement or primers. However, these stages must repeat one sort or two or more sorts of arrangement amplified as enough to connect over sufficient number of times for that of production To shake . In flush end connection, one sort or two or more sorts of high-concentration arrangement, one sort, or two or more sorts of cloning vectors are needed rather than a case of adhesion end connection. This ligation must become as [be / they / T4 ligase, E, and Cori (ELcoli) ligase], and it must perform it in existence of gauze. If amplified output is obtained, a connecting process method will be performed to a person skilled in the art by the standard processing method for using well-known conditions. A cloning method which does not use flush end ligation controls directivity or multiplex nature of insertion to a cloning vector of amplified output. This process can be used for in vitro variation indecement. An oligonucleotide primer does not need to be correctly complementary in nucleic acid arrangement which should be amplified. They only need to hybridize in arrangement 10 minutes which should be elongated with thermostable enzyme. Since output of an amplification reaction when a primer used is not correctly complementary to the original mold has not mold arrangement but the arrangement of a primer, in vitro mutation is introduced. In a continuing cycle, since this mutation does not need un-opposite stand brimming any more, as for #I Radiation , it is carried out at efficiency which is not limited. Thus, a produced mutant can be inserted in a suitable vector by standard biological technique, and the mutant characteristic like production voice of firefly white matter changed into this vector can be given. A process of making the changed above DNA arrangement is repeatable to DNA changed using a different primer for inducing change of other arrangement. Thus, a series of variation arrangement is produced gradually, and although each thing newly added to this series does not serve as small deer powder from a front thing, it can differ at a remarkable point from the original source arrangement of DNA. Thus, since a very un-proper primer cannot be operated, finally in a single stage, change which is not obtained can be performed. When sequence with sufficient quantity of a primer complementary to a chain which should be amplified is contained, 0 to which the primer can have un-complementary arrangement in a part of the arrangement -- for example (A promotor, a linker coding sequence, etc.) a nucleotide sequence which is not complementary in mold arrangement -- a primer -- on the other hand -- or it can be made to be able to combine with both 5' ends, and can add to output of an amplification process by this. After an extension primer is added, only sufficient number of times repeats a process and it obtains the new mold containing an un-complementary nucleotide insert portion in quantity [ Desire ]. fragmentation comparatively combined by this for a short time (for example, 2 hours or less) using a simple technique -- abundant -- production To shake -- things are made. Specific nucleic acid arrangement about infection, a genetic disease, or a cellularity disease, for example, cancer, for example, detection of tumor genes, and the feature-ization can be performed using this method. When there is very little quantity of nucleic acid which can be used for analysis, it is useful at a prenatal diagnosis of sickle cell anemia using DNA obtained, for example from an embryonal cell. This amplifying method is especially useful, when analyzing a small-quantity sample using an analysis method which uses a nonradioactive detection method and which originally is not sharp, or when it is a case where a radiometric method is used and prompt detection is desired. It is related with the object of the present invention and a genetic disease includes specific deficiency of genome DNA from living bodies, such as sickle cell anemia, alpha- Thalassemia , and beta- Thalassemia , and/or variation indecement, for example. sickle cell anemia -- this method - Ri -- oligomer restriction analysis given in European patent laying-open of application 164th of December 11, 1985 issue after amplifying suitable Ri NA arrangement, and No. 054 specification -- or RFLP Mr. analysis, It can detect easily, and an absence of a multi-form restriction site which related to a variation part which causes this disease closely can detect alpha- Thalassemia , and existence of that restriction site can detect beta- Thalassemia . All these genetically determined diseases are detectable by amplifying suitable arrangement, and analyzing it, without using a radioactive probe with a Southern method. In such a method, a small amount of samples of DNA from Amniotic fluid including for example very low level arrangement [ Desire ] are amplified, and it cuts by a restriction enzyme, and analyzes with a Southern method. Use of a nonradioactive probe becomes easy with an amplified high level signal. After amplifying a small-quantity sample of DNA even to the usual level in another mode, It carries out by continuing a cycle of an extension reaction, and introduces directly a nucleotide derivative detected in this case easily (it is (like a ffZ p-sign or nucleotide triphosphate which carried out the biotin sign)) into the last DNA output, It is possible to analyze this by restriction enzyme processing and separation by an electrophoresis method, or other suitable methods. In another mode, nucleic acid can be exposed to restriction and nuclease specific before amplification. That fragmentation amplified in spite of having carried out restriction enzyme processing of the DNA sample beforehand appears since cut arrangement cannot be amplified suggests an absence of a part of nuclease in amplified arrangement. Amplified existence of arrangement or an absence is detectable by a suitable method. Practical application of a method of the present invention is this specification, the European patent No. 164, 054 specification (same as the above), and Bio/ Technology of Psyche and others (Saiki), It can illustrate by the use for making detection of sickle cell anemia easy by three volumes and the oligomer restricting method indicated to 1008~1012 pages. Sickle cell anemia is a hemoglobin disease which happens by change of only one base pair in the 6th codon of beta-globulin gene. The method of the present invention can do Stop used also for detecting directly change of a little salt machine pair of nucleic acid (it is (like genome DNA)) arrangement using an arrangement specific oligonucleotide. In this method, arrangement change which arises from cancer, infection, or a genetically determined disease, for example, heritable deletion, is directly detectable, and if it is original, the necessity for required restriction enzyme digestion, electrophoresis, and gel operation is lost. If an arrangement specific oligonucleotide is used in a A pot plot method after amplification written in this specification, singularity and susceptibility of a probe can improve and it can obtain a signal which can be interpreted within 6 hours using a 0.04-g sample. If quantity of a sample spotted on a film is increased to 0.1-0.5 g, an oligonucleotide by which the nonradioactive label was carried out can be used instead of a radioactive probe used by a conventional method. An arrangement specific oligonucleotide where a size can apply to use of less than 19-Maher's arrangement specific oligonucleotide therefore which has discriminating power further can be used for a method given in the following. Although RFLP needs a multi-form restriction site about this disease about a genetically determined disease, An arrangement specific oligonucleotide detects heritable deletion directly, and is very useful by mutation of a base of single What to sick analysis like To live hemoglobin C Disease , alpha-1-anti trypsin, and beta- Thalassemia . Since a genetic variation object showing a different allele (for example, HLA type) is discriminable using an oligonucleotide, the possibility of a kit on the basis of an arrangement specific oligonucleotide containing thermostable enzyme is suggested. When it is going to detect arrangement change of a nucleotide in one mode of the present invention, As mentioned above, a sample amplified using one primer about each chain of each nucleic acid presumed to include nucleotide change is directly spotted on a series of films, and it cheats out of an arrangement specific oligonucleotide probe and hybridization which are different in each film and by which the sign was carried out. A method of spotting a sample to a film is indicated in Kahotos (Kafotos) et al., seven Nucleic Ac1ds Re5earch, 1541~1552 pages, and 1979. It can pretreat, before adding a probe using blur hybridization solution which contains dodecyl sodium sulfate, ficoll (Ficoll), serum albumin, and various kinds of salt for a DNA sample fixed to a film, if it summarizes and says. Subsequently, a thing specific to each arrangement change which is the nucleotide probe by which the sign was carried out and it is going to detect is added to pre hybridization solution and same hybridization solution. In - boat fishing which applies this hybridization solution to a film and gives this film to hybridization conditions which change with a model of a probe, a model of length and an ingredient, concentration, etc., a hybridization -- about 25~75degreeC preferably 35~65degreeC, and 0.25~50 hours -- desirable, it carries out for less than 3 hours. As stringency of conditions becomes large, complementarity required for a hybridization of a probe and a sample becomes larger. If a background level is high, stringency will become high so much. Stringency conditions can be taken also into washing. Washing removal of the probe by which a hybridization is not carried out using a suitable means is carried out from a sample after a hybridization. Although washed once or more ~ time during about 10 minutes by 25~75 degreeC with EDTA of standard salt phosphoric acid EDTA (SSPE) lPO (Macl [of 180mM], Na of 10mM) 4, and IM of various concentration, and pH 7 and 4 solution, this time changes with temperature -- it ranks next zero and detects this sign using the suitable detecting method. An arrangement specific oligonucleotide used here is an oligonucleotide prepared and chosen by an above-mentioned method in -G in order to prepare a primer. As mentioned above, the arrangement specific oligonucleotide must include a field of arrangement covering nucleotide change which should be detected, and must be specific to nucleotide change which should be detected. If it is a request to detect whether a sample includes variation of curdy red corpuscle anemia, An oligonucleotide containing a nucleotide sequence part characteristic of a normal beta-grotesque pudding gene is prepared, and also an oligonucleotide including a nucleotide sequence characteristic of a sickle erythrocyte allele is prepared. Each oligonucleotide is made to hybridize with a reproduction of the same sample, and it is determined whether a sample includes variation. Since arrangement which is carrying out office In and was saved to a specific field of a first exon faces across a polymorphism field of a HLA class ■ gene, a complementary oligonucleotide primer can be prepared to a chain with which 5' where a first exon was saved, and 3' end counter. The number of oligonucleotides used for detection of a polymorphism field of a HLA class ■ gene, It changes with models of a gene which has cluster structure or has a field of base pair variation currently distributed scatteringly. Like [in ILA -D low alpha], when this field has cluster structure, one oligonucleotide is used about each allele. While this field is distributing like [in HLA -D low beta and HLA - DR-beta], what is many probes from one sort about each allele, and includes an allelic variation object, respectively is used. in I (LA - DQ-beta -- and) ILA -Di?-beta, three sorts of probes are used about three fields of gene loci where an allelic variation breaks out. detection of insulin dependence genuineness diabetes (IDDM) -- the [HLA -DRbeta] -- a probe of the four 2 second Be exotic is used. Hub Rotary can be directly presumed by analysis of a DNA sample of an individual by separation (segregation) in a family to a case. Combination ( Halo type ) of a specific allele of arrangement specific oligonucleotide reactivity can be identified in the Peter junction cell by using restriction enzyme digestion of genome DNA before amplification. For example, when subfield A, B, and C in which variation is possible is highly found out in an amplifying region of single What by DQbeta, And combination A. I. Artificial Intelligence of hub Rotary possible when there is six different arrangement (A. I. Artificial Intelligence-6, B1-6. Cl-6) in each field, B-2, C1;^1, B-2 *C4; A2, B-2, C1; A2, B-2, C4; aluminum, B3, C1; aluminum. In DQbeta seat, it is an individual with B3, C4;aluminum, 82, C1i and A. I. Artificial Intelligence, B-2, and C4 by arrangement specific oligonucleotide probe analysis At and A2 ; B-2, B3 ; A type division can be carried out as a thing containing ClC4. When genome DNA is digested by polymorphism Limiting enzyme before amplification, and when this enzyme cuts an allele between primers both, there is no reactivity with an arrangement specific probe because of lack of amplification, and it does not provide equivalent information. When this enzyme does not cut an allele, a result in genome DNA which is not digested and digested becomes the same, and, as for that result, it does not provide equivalent information. When this enzyme cuts only one allele, both hub Rotary can be presumed by comparing a probe reactivity pattern of DNA which is not digested and digested. 1~ by which these hub Rotary are uncut genome DNA and many forms, and recognizing a part between primers is known -- it can presume by comparing the reactivity of genome DNA cut with some enzyme, and an arrangement specific oligonucleotide. A specific target molecule from which the length of an arrangement specific oligonucleotide should be detected, In the case of the 0 present invention which changes with factors of many like a source of an oligonucleotide, and nucleotide composition, typically, although an arrangement specific oligonucleotide has 15~25 nucleotide, it may contain many or few nucleotides. Since a That rough single mismatch destabilizes more less than one probe which is for example, 16-Maher 19 Ma although an oligonucleotide whose length is at least 197 - improves singularity and/or susceptibility, still higher arrangement specific discriminating power can be shown. Since singularity increases by amplification, longer length may not be indispensable, said [of a hybridization and the washing temperature] may be carried out, they may be low about salt concentration, and it is preferred that length uses an oligonucleotide of I9 Maher sheep slot. When putting a sample on a film first and detecting by an oligonucleotide subsequently, the sign of this oligonucleotide must be carried out with photochemical [like a spectrum], biochemical, and a suitable immunization study Marking remnant machine detectable by a chemical means. There is an antibody which can form an oligonucleotide and a complex under suitable conditions in an immunization study means, 0 which has polypeptide or lectin which can form an oligonucleotide and a complex under suitable conditions in a biochemical means -- for exampleA fluorescence dose, an electron Dance (electron-dense) reagent, There is a radioactive label or biotin like enzyme which an insoluble reaction product can be settled or can be detected by coloring, for example, horseradish peroxidase, a thing like alkaline phosphatase, and izp. When using biotin, it is made to combine with an oligonucleotide using a spacer arm. Horseradish peroxidase is preferred as a Marking remnant machine. In a "contrary" A pot plot method, a sign is carried out with a sign which can detect at least one of at least one piece and/or four sorts of the nucleotide triphosphate of a primer, and the account of a sign of the amplified arrangement to generate is carried out. These Marking remnant machines may exist in a reaction mixture from the beginning, or, in addition, can introduce a sign into amplification output in a second-half cycle of amplification. Next, when arrangement variation (either normal or a mutant) exists, A sample amplified by zero-order [which spots an arrangement specific oligonucleotide of a non-sign which can carry out hybrid formation in amplified nucleic acid arrangement on a film under the above pre hybridization conditions (or fixation) ] is added to a film pretreated under hybridization conditions as mentioned above. Finally, a detection means is used, and when arrangement by which a nucleic acid sample was amplified carries out a hybridization to an oligonucleotide fixed to a film, it detects. A hybridization happens, only when arrangement combined with a film including variation exists in amplification output, namely, only when complementary to a field of arrangement to which arrangement of a probe was amplified. In another mode of a "contrary" A pot plot method, The above "not using a sign, it amplifies like [in an order J dot block method], Amplified nucleic acid arrangement which contains it when variation exists, and an arrangement specific oligonucleotide probe which can be hybridized and by which the sign was carried out, A sample amplified by zero-order [which is spotted on a film under the above pre hybridization conditions (or fixation) ] is added to a film pretreated under the above hybridization conditions. Subsequently, an oligonucleotide which carried out the sign, or its fragmentation is made to separate from a film, and when amplified arrangement in a sample carries out a hybridization to an oligonucleotide by which the sign was carried out, a detection means is used and it enables it to detect it. Isolation can be caused by adding a restriction enzyme to a film which recognizes a restriction site of a probe, for example. This method known as oligomer restrictions is written still in detail in the European patent laying-open-of-application No. 164, 054 specification of December 11, 1985 issue. A genetically determined disease by which reverse A pot plot either order or a method is detected from one of living things includes substitution in one of mutation or many forms of a base pair of nucleic acid like specific deficiency, insertion, and/or genome DNA. As an example of a disease with which variation of a base pair is known, there are sickle cell anemia, hemoglobin C Disease , alpha- Thalassemia , beta- Thalassemia , etc. There are a cancerous disease and infection containing RAA tumor genes, for example, n-RAS tumor genes, in other detectable illnesses. The A pot plotting method can also be used for a transplant of an organization and HLA typing in the field of determination of the ease of falling ill, and paternity with illness. A HLA class ■ gene consists of alpha of HLA-DR, HLA-DQ, and HLA -DP territory fields, and a beta gene, its polymorphism is very high, and genetic complexity in those DNA levels is more remarkably [than many forms generally defined by serological typing] large. A method of the present invention can be used for detecting four DNA arrangement which codes a HLA class ■beta protein prize (for example, DRbeta) about insulin dependence genuineness diabetes (IDDM). Four sorts of DNA arrangement about IDDH if it says simply, (1) 5 '-GAGCTGCGTAAGTCTGAG-3' (2) 5 '-GAGGAGTTCCTGCGCTTC-3' (a) 5 -- '-CCTGTCGCCGAGTCCTGG-3' -- and (4") 5 -- it is chosen from a group which becomes '-GACATCCTGGAAGACGAGAGA-3' or them from NA chain in Complementary . An arrangement specific probe which carries out hybrid formation can be prepared to one sort of these arrangement, or two or more sorts. Various infection student diseases can be diagnosed when specific DNA arrangement characteristic of a reason microorganism exists in a clinical sample. There is a parasite like a plasmodium leading to salmonella, chlamydia, bacteria like neisseria, a virus like a hepatitis virus, and malaria in these reason microorganisms. As for May 13, 1986 published by Falco and others (Falkow), United States patent reexamination certificate pipe No. 814.358. 535 has indicated use of a specific DNA hybridization probe to diagnosis of a susceptibility disease. comparatively -- a small number of illness -- primeval -- since a thing exists in bt floor sample from an infected patient, DNA extracted from these may constitute only very small Fractionation of the total DNA in a sample. Conventional sensitivity and singularity of a method can be remarkably improved by amplifying arrangement presumed specifically and carrying out hybridization detection before fixation of a DNA sample. It writes in the European patent No. 63, 879 specification of word (Ward), If a probe by which the nonradioactive label was carried out can be used, color-enhancing enzyme which will be simplified considerably and which combined a biotin content DNA probe with a red-throated loon gin or biotin specific antibody in this method will detect everyday clinical use of a DNA probe for diagnosis of a susceptibility disease. Although this type of the detecting method is convenient, Facilities and sensitivity which are taken to make a method of word (Ward) useful to an everyday clinical examination with Falco (Palkow) with combination of use of a probe by which the sign was carried out to specific DNA amplification and stability by a method of the 0 present invention whose susceptibility is comparatively low are obtained. Concrete use of this amplification art over detection and surveillance of an AIDS virus is explained below. It is a primer and a probe, and it is substantially saved in ° nucleic acid of an AIDS virus, respectively, and what is designed to amplify and detect specific nucleic acid arrangement to nucleic acid in a A IOS virus is used for amplification and the detecting method. Therefore, arrangement detected is complementary enough to nucleic acid in an AIDS virus, is existence of enzyme and nucleotide triphosphate, and must start a polymerization at room temperature preferably. This probe is a biotin-ized probe which biotin has combined with a spacer arm which has formula % type % (Y is O, NH, or N-CHo among a formula, X is the number of 1~4, and y is the number of 2~4). This spacer arm is combined with a Psoralasin residue machine of a formula again. Like [this Psoralasin residue machine / of cola Gitebe et al. (courage-Tebbe), Biochim, Bio h s, Ac nucleic acid, 697 volumes, 1982, and a 1~5-page statement], "Into a Gapped circle (gapped circle) J probe, intercalation is carried out, it is crosslinking, and a chain hype Redisation field has covered a field included in a primer in the 1 end of a Gapped circle. Details of biotin-izing and the A pot plotting method are written still more completely in the United States patent No. 4.582. 789 specification of April 15, 1986 issue, and a U. S. Pat. No. 4, 617.261 specification of October 14, 1986 issue. Using this amplifying method, DNA is made to produce in sufficient quantity from a single copy human gene, and it can use for detection of DNA directly, simple nonspecific DNA dyeing agent, for example, ethidium bromide. In addition to detection of a susceptibility disease in a genome of a living thing, and pathological abnormalities, it can also use for detecting DNA many forms without a morbid state. If it summarizes, the amplifying method will provide a method of amplifying one or more sorts of specific nucleic acid arrangement which uses a chain reaction and thermostable enzyme, will make primer extension output produce in the above-mentioned reaction, and will enable it to commit this as a mold of a subsequent primer extension reaction next. This method is useful especially although nucleic acid arrangement which exists only in a small quantity extremely at first is detected. The following examples are provided only for illustration and it does not mean limiting a range and a claim of an invention. In these examples, unless all percentages in particular refuse, in the case of weight % and a fluid, it is capacity % and, in the case of a solid, Centigrade expresses all the temperature. composition of Anus and a primer -- the following two kinds of primers were prepared by a following method. Each of these primers is 20-Maher, sets an interval of 110 base pairs to a chain with which genome DNA faces, and Annealing it at those 5' ends to it. A and Diethyl phospho lamination Daito compounded in accordance with a method of automated synthetic methods Beaucage and Caruthers (TeLrahedronLetters22 (1981) : 1859-1862), a controlled hole which was derivative-ized by a nucleotide -- it was made to condense one by one on a glass base material of sizeas formation of Evacuation trityl according [this method] to trichloroacetic acid in dichloromethane, and an activation proton donor -- benzo-- doria -- capping by condensation which uses Sole , a tetrahydro franc, an acetic anhydride in pyridine, and dimethylamino pyridine is included. Cycle time was for about 30 minutes. yield in each stage is essentially quantitive, and recovery and a spectroscopic analysis of dimethoxytrityl alcohol which are emitted during formation of Evacuation trityl determined it. Deprotection and a refining solid base material of B and oligo deoxyribonucleotide were taken out from a column, and it was exposed to dark ammonium hydroxide of 1 A at room temperature in a sealing tube for 4 hours. Next, solution containing an oligo deoxy nucleotide which removed a base material by filtration and was protected partially was set to 5-hour 55-degreeC. Ammonia was removed and it was valid for polyacrylamide gel for preparative isolation in a residual substance. Electronic migration was performed for 90 minutes in 30 V/all, and a band which contains output next was identified by UV shadow of a fluorescence plate. A band was cut down and it was eluted in 4 degreeC with distilled water of 11R1 overnight. This solution was applied to a RP -HPLC column, and it was eluted by a gradient of 7~13% of acetonitrile in 1% ammonium acetate buffer solution (pH 6, 0). This elution was monitored by UV absorption by 260 nm, and suitable halving was collected, and it quantified by UV absorption in constant capacity, and evaporated to dryness at room temperature by a vacuum centrifugal in a plane one. It is '''P with the feature of C and an oligo deoxy nucleotide by polynucleotide kinase and T A "P-ATP about a refined examination aliquot of an oligonucleotide. - The label was carried out. A compound by which the label was carried out was examined by autoradiography of 14-20% polyacrylamide gel after electrophoresis for 45 minutes in 5 Qv/an. A molecular weight is checked by this method. Digestion to a nucleoside of an oligo deoxy nucleotide according base composition to snake venom Gestalase and bacteria alkaline phosphatase, and this -- Continuing -- < -- separation and a fixed quantity of the nucleoside using a negative phase HPLC column and a 10% acetonitrile+1% ammonium acetate mobile phase determined. ■ The amount genome DAN of isolation polymers of human genome DNA from cellular in, It essentially [Maniatis etc. ] followed to the above-shown and P2S5-281, and was isolated from T@ A cow system of homozygosity about a normal beta-grotesque bottle available as 1J2219c from a human Genetic mutant cell Devoji tree, a cam ten, and NJ. ■ Thermes Aquaticus (refining American type culture collection of polymerase from Thermus main Inhibition I± Animal ( Translocation ) -) The following carries out culture-medium Te multiplication of the 12301 park round live, Rockville, and the Thermes Aquaticus (Thermus... uaticus) YTI stock that can be obtained from MD that there is no control in any way as ATCC A disease 25.104 in a flask. Sodium-acid-citrate 1wM potassium phosphate pH7, 95 a+M ammonium chloride 10mM magnesium sulfate 0.2mM calcium chloride 0. In+M sodium chloride Ig/l yeast extract Ig/l Trypton Ig/l glucoseIt is a flask mother stock which cultivated 2-g/l ferrous sulfate 0.01mM (it is p before autoclave) I in 70 degreeC in the adjustment above-mentioned culture medium to 8.0 overnight! 10 A fermentation tub was inoculated. A mother stock of a total of 600 - of mother stock flasks was inoculated into the same culture medium of 101. pH was Chairperson (ed) to 8 and 0 with ammonium hydroxide, dissolved oxygen was adjusted to 40%, and temperature was adjusted to 70'C, and churning speed was 400 rpm. The Kaledin etc. performed all 66 steps refined using a method of being different in the 6th step in 4 degreeC after multiplication of a cell using a method (adding change slightly) of above-shown , about five steps of the beginning. Fractionation speed in a column was considered as the time of 0.5 power rum /, and capacity of a gradient under elution was made into 10 capacity of column. The postscript of a desirable method of replacing with this is carried out to Example 6. If it summarizes, they will be T of the above-mentioned cultivation, and the Aqua typhoid fever (L Happy birthday in cell concentration of 1.4g of second-half logarithmic phase dry weight / l, a cell was Harvest (ed) by centrifugal separation after cultivation for 9 hours.). the inside of buffer solution 8O- which contains 50 mM Tris-HCl (pH 7, 5) and Oll mM EDTA for a 20-g cell -- having been suspended. A cell was dissolved and centrifugality of the dissolution thing was carried out in 35. OOOrpm in a rotor of 4 degreeC for 2 hours. Top Qing dynasty are collected (drawing part A) and parts for protein drawing which precipitates between 45~75 saturation of ammonium sulfate are collected, It dissolved into buffer solution containing 0.2M potassium phosphate (p) 16.5, 10mM2-mercaptoethanol, and 5% glycerin, and it dialyzed to the same buffer solution at the end, and obtained drawing part B. Next he wanted to apply drawing part B to the 2.2X30-cm column of DEAE - cellulose balance-ized with the above-mentioned buffer solution, parts for drawing which wash this column with the same buffer solution, and contain protein (absorption at 280 nm determines) were collected. - A clue was used, a part for It is lid white matter drawing was dialyzed to the second buffer solution containing 0.01M potassium phosphate (pH 7, 5), 10IIIM2-mercaptoethanol, and 5% glycerin, and drawing part C was obtained. 100 minute C was applied to the 2.6X21-cm column of a hydroxy apatite balance-ized with the second buffer solution. Next, enzyme was eluted by a straight line gradient of 0 and 01~0.5M potassium phosphate (pH 7, 5) buffer solution which washes this column and contains 10mM2-mercaptoethanol and 5% glycerin. Halving (90~180mM potassium phosphate) containing DNA polymerase activity was collected, and it condensed 4 times using an Amicon churning cell and n+10Jl!, and it dialyzed to the second buffer solution, and obtained drawing understanding. It applied drawing understanding to a 1.6X2Bcm column of DEAE-cellulose balance-ized with the second buffer solution. zero drawing which washed this column and was eluted in DNA polymerase by a straight line gradient of 0.01in second buffer solution~0.5M potassium phosphate -- and it measured about contamination of nuclease and exonuclease -- alike -- havingChange of a molecular weight of phage lambdaDNA after incubating with superfluous DNA polymerase, or super coil plasmid DNA is detected by electrophoresis (end nuclease), And DNA was performed after processing by a restriction enzyme which makes some fragmentation cleave (exonuclease). Only a part for DNA polymerase drawing which has the minimum nuclease contamination (65~95InM potassium phosphate) was pooled. Gelatin which carried out autoclave to this pool was added in quantity of 250n / -, and it dialyzed to the second buffer solution, and obtained drawing part E. Drawing part E was applied to a phospho glucose column, and it was eluted by gradient 100- of 4 M KCl among [0.01~0] 201IIM potassium phosphate (pH 7, 5). It halved as mentioned above and measured [contamination / of nuclease / exonuclease] about polymerase activity further (methods, such as Kaledin). Pooled halving was dialyzed to the second Heel liquid and then it condensed by dialysis to glycerin and the second buffer solution 50%. 5 OS-PAGE determined a molecular weight of polymerase. Marker protein was phosphate B (92, 000), bovine serum albumin (66, 200), ovale Bumin (45, 000), carbo Nickian hydrase (a1, 000), soybean trypsin inhibitor (21, 500), and a lysozyme (14, 400). Preliminary data was not 62, 000~63, 000 Dalton reported to articles (for example, Kaledin etc.) but about 86, 000~90.000 Dalton. It is this polymerase! 25 mM Tris-HCj (pH 6, 4, pH 8, 0) O side, the LM K side, 10mM MgCl!, z and 1mFI2-mercaptoethanol, and every 10 nmole dGTP. It incubated in mixture 5OJd containing polymerase of dATP and TTP, 0.5-microcurie ('H) dCTP, "cow activated" thymus DNA of 8 n, and 0.5-5 unit. It digests by DNase I and DNA "activated" is a natural manufacture thing of DNA after carrying out partial hydrolysis until 5% of DNAs move to a part for acid-solubility drawing. You performed this reaction for 30 minutes in 70 degreeC, and made it stop by addition of 50 m of saturation aqueous conclusion containing 0.125 M EDTA-Naz of sodium phosphate. It is indicated to the above-shown, and Kaledin etc. made and processed a sample, and it measured activity. As for this result, polymerase showed that it was more active than an active half in pH 8 and 0 in pH 6 and 4. On the other hand, Kaledin found out that oxygen had active 8% in pH 8 and 3 in pH about 7 and 0. Therefore, pH profile of thermostable enzyme of the present invention is larger than that of enzyme, such as Kaledin. Finally, when one kind or two or more kinds of nucleotide triphosphate was removed from a DNA polymerase measurement reaction mixture, activity was hardly observed using enzyme of the present invention, activity was in agreement with an anticipation value, and enzyme showed high faithfulness. On the other hand, observation Now activity of Kaledin does not correspond with an anticipation value using the above-shown, but introduction in which nucleotide triphosphate made a mistake is suggested. ■ It is genome DNA of lu of the amplification reaction above, 25+nM Tris-1 (J (p) 18.0) 50mM KCl, 10 n+M gate g (/!□, 5IIIM dithiothreitol) It diluted in initial water reaction capacity of 10011I containing primer PCO4, 1 and 5mMdATP of 2 or 1 primer PCO of 2001 tg/d gelatin and 1 Oh , 1.5mM dcTP, 1.5mM dGTP, and 1.5mMTTP. Heated a sample for 10 minutes in 98 degreeC, genome DNA was made to denature, and then it cooled to room temperature. Polymerase 4I from Thermes Aquaticus was added to a reaction mixture, and mineral oil of Loojll was stratified. Next, a sample was put into the above-mentioned fluid handling and an aluminum heating block of a heating device using a thermostat for a pipette programmed in order to supply a Melting object, and varying temperature. The following program cycle was repeated and a DNA sample was applied to amplification of 20 cycles. 1) Heat from 37degreeC to 98 degreeC over 2.5 minutes in a heating block, cool from 98degreeC to 37 degreeC over [; and 2] 3 minutes, and make Annealing of a primer and D N A possible. After the last cycle, a sample is incubated by 55 degreeduring 10 more minutes C, and the last extension reaction is completed. ■ composition of an oligo deoxyribonucleotide probe, and phosphorylation -- DNAN Robof which has following arrangement: and it calls R324 and by which the label was carried out was prepared. * shows a label during arrangement. This probe has the length of 40 bases, and contains from the 4th codon of a gene to the 7th codon, and is complementary in a normal beta grotesque bottle allele (betaA). Relation between a primer and a probe is shown typically below. It is [T A "P-ATP (about 7000 Ci (s) /mmo le) of T4 polynucleotide kinase of four units, and about 40 pmole (s), and] 70mMTris (pH 7, 6) about a probe of a 20pmole which compounded this probe in accordance with a method indicated to example 1. Work , 10mM Mg (you made it contact for 60 minutes in 37 degreeC in reaction capacity of 401 containing J z, 1.5mM Spermine , and 10mM dithiothreitol) Next, the amount of whole picture is prepared to 100IJ1 by 25mM EDTA, and it is Tris -EDTA 11 Rinse (10mM Tris). (TE) Maniatis etc. were refined in accordance with the above-shown and a method of p466467 on a l ml spin dialysis column balance-ized by 0 and 1 mM EDTA 5pH 8 and 0. TCA precipitation of a reaction product -- R524 -- a ratio -- activity is 4.3 u Ci/pmole and it was shown that the last concentration is 0.118pmole/ l. ■ Sample 4p! amplified from A pot plot hybridization above-mentioned ■, and suitable dilution things 5 and 6I of beta-grotesque bottle plasmid DNA calculated so that 70, 75, 80, 85, 90, 95, and amplification efficiency of 100% might be represented -- 0.4 of 200 ill -- carrying out NNaOH (ing) and 25 mM EDTA T:m Interpretation And it spotted on a nylon filter. This wets a filter with water and it puts this in a device for dot block manufacture which holds a filter in a fixed position, A sample is applied and it is Reed and Mann, Nucleic Ac1dsResearch, and 13.7202. It is made to be indicated by 722H1985. Each Well was rinsed by Q (a, 6MNaCl % 200TIIM Na) 20xsspE IzPO4 of l ml, and 20+nM EDTA. Next, a filter is taken out, and it washes in 2OX 5SPE, and is among vacuum open, and heated for 30 minutes in 80 degreeC. They are 3XSSPE and 5x Denhardt solution (I X =0.02% polyvinyl pyrrolidone) after heating and about each filter. Hybridization solution 16m which comprises 0.02% ficoll, 0.02% bovine-serum-albumin, 0.2mM Tris, 0.2mM EDTA, and pH 8, 0, and 0.5%SO8 and 30% Holm amide was made to contact, and it incubated in 42 degreeC for 2 hours. Next, probe 1ls24 of 2p11ole was added to hybridization solution, and a filter was incubated for 2 minutes in 42 degreeC. At the end, room temperature washed each hybridized filter for 10 minutes twice by 2 X5SPE and 0.1%SDS of 100+d. Next, a filter was processed for 10 minutes by 60 degreeC by 2 X5SPE of 100-, and 0.1%SOS. Next, autoradiograph processing of each filter was carried out. A signal was compared with the standard serial dilution beta-grotesque bottle manufacture things which analyzed an autoradiogram of a A pot plot 6■ which appeared easily 2 hours afterward, and 2 hours after examination of an autoradiogram, and were prepared by Haem/□I-digestive pBR:beta ^ about intensity. betaA is a wild nature type allele set and indicated to the 5cience % above-shown, such as 5aiki. It is shown by analysis of a reaction product that the whole amplification efficiency is about 95%, and this is equivalent to a 630, 000 times as much increase as a beta-grotesque bottle target sequence. They are two samples of one codfish of genome DNA extracted from Mo1t4 cellular in as indicated for example 1 of 16 amplification reactions, respectively 50mM KCl and 25mM Tr'+5-HCl2 18.0, 10mM MgCjl!z Gelatin of primer PCO4, 200 codfish / - of primer PCO3 of 1 ti?'+, and 1-, 10% dimethyl sulfoxide, and every 1.51 dATP. It diluted in reaction capacity of 100IJI containing dCTP, dGTP, and TTP. After heating this mixture for 10 minutes by 98 degreeC and denaturing genome DNA, a sample was cooled to room temperature and polymerase 4 pl from Thermes Aquaticus indicated for Example 1 was added to each sample. Mineral oil was stratified to a sample and condensation and an evaporation loss were prevented. One of the samples was put into a heating block of machinery indicated for Example 1, and the following program cycle was repeated, and it applied to a reprint of 25 cycles. (1) By heating from 37degreeC to 93 degreeC over 2.5 minutes, and cooling from 93degreeC to 37 degreeC over [: (2) ] 3 minutes, the anil of a primer and the DNA was carried out, and it held for 2 minutes to; and (a) 37-degreeC. After the last cycle, a sample was incubated for 10 more minutes in 60 degreeC, and the last extension reaction was completed. The second sample was put into a heat conduction container of a temperature cycle (heating and cooling) opportunity. It is attached to a microprocessor which controls automatically timing of the Belle Tyr heat pump, an amplification cycle, a temperature level, a temperature gradient, and temperature which adjusts temperature to a heat conduction container at the upper part or a lower part in this machinery. The second sample was given to amplification of 25 cycles which repeats the following program cycle. (1) Heat from 37degreeC to 95 degreeC over 3 minutes, hold for 0.5 minute to; (2) 95-degreeC, produce denaturation, cool from 95degreeC to 37 degreeC over [; (a) ] 1 minute, and hold for 1 minute in; and (4) 37-degreeC. ■ Two examinations were done for assay analysis, i. e., A pot plot analysis, and agarose analysis. For A pot plot analysis, make of DNA Blow * of next arrangement:5'-"CTCCTGAGGAGAAGTCTGC-3' (RS18) by which the label was carried out was carried out 8 round. * shows a label during this arrangement. this probe has the length of 19 bases -- the [of a gene] -- it is complementary to a normal beta-grotesque bottle allele (betaA), including the 4~7th codon. A relation of an outline of a primer and a probe is shown below. A probe of The margin was compounded below by a method indicated to example 1.1. With T A "P-ATP (about 7000 Ci (s) /rarao l e) of 4 unit T4 polynucleotide kinase and about 40 pmole (s), it is about a probe of 10p Mongolia ole, 70 mM Tris-H (J (pH 7, 6), 10mM Mg (you made it contact for 60 minutes in 37 degreeC in reaction capacity of 40Jll containing 42, 1, F++M Spermine , and 101 dithiothreitol)) Next, 25mM EDTA adjusts the amount of whole picture to 100J, and it is Tris -EDTA (TE) buffer solution (10mM Tris-HCA'.). Maniatis etc. were refined in accordance with a method of the above-shown and p A 66-467 on a l ml spin dialysis column balance-ized by 0, 1 mM EDTA, pH 8, and 0. TCA precipitation of a reaction product -- R51B -- a ratio -- activity is 4.6 p Ci/pmole -- and the maximum P and concentration -- 0.114 pmole/mu! It came out and a certain thing was shown. It is [sample 5micro! amplified from the 1., and] E instead of thermostable enzyme, About sample 5I which a Klenow fragment of the Cori DNA polymerase ■ was used, and also was amplified like the method, they are 0.4NNail (it diluted by 25vM EDTA, and spotted on a nylon filter of two sheets.) of 195I. In this case, a filter is wetted with water first and a filter is put into a device for Tomb lot manufacture held at a suitable place, As it is valid in a sample and is indicated more by Reed, Mann, and the above-shown, it is 20 X SSP [of 0.4 ad (a, 6M NafJ) !6 which rinsed each Well by 200 nucleic acid MNaHtPO* and 20 a+M EDTA, next a filter were taken out, and it heated for 30 minutes in 80 degreeC in vacuum oven. They are 5 X5SPE and 5x Denhardt solution (1x = 0.02% polyvinyl pyrrolidone) after heating and about each filter. 0.02% ficoll, 0.02% bovine serum albumin, and 0.2ynM Tris, 0.2mM EDTA, pH 8, 0, and hybridization solution 6- that comprises SDS 0.5% are made to contact. And to zero-order [which incubated for 60 minutes in 55 degreeC], probe R318 of 5u1 was added to hybridization solution, and a filter was incubated for 60 minutes in 55 degreeC. A filter was processed by 5x5SPE of 2 more times (1 time for [1 minute] and the 2nd time for 3 minutes) 100M1, and 0.1%SDS to zero-order [which washed each hybridized filter for 10 minutes at room temperature twice by 100-d 2XSSPE and 0.1%SOS at the end]. Next, autoradiograph processing of each filter was carried out, and a signal appeared easily 90 minutes afterward. In agarose gel analysis, it is I XTBE 71 Rinse (0, 089M Tris) about an every 5 tII amplification reaction mixture. 0.089M?1lll! It loaded on acid, 4%NuSievein 2 mM EDTA10, and 5% agarose gel, and carried out electrophoresis for 60 minutes in 100V. After dyeing by an ethidium bromide, DNA was visualized by UV firefly point. This result showed that machinery used in Example 1 and this example were similarly effective in amplification of DNA, and showed high intensity 110 base pair with similar separate intensity corresponding to arrangement of the object, and other separate bands of a small number of very low intensity. On the other hand, an amplification method which transports a reagent after each cycle using E and the Freneau fragmentation of collie polymerase I brought about Yo time (seer) of DNA which arises from nonspecific amplification of many unrelated DNA arrangement. FILA-DQ, DI? And in evaluation of DP pathological change, it is expected that amplification and the same improvement of detection will be attained. In the above-mentioned experiment, amplification reaction buffer is 10+aMMgCj!When it contains each every 150~200 voice [instead of not x but 2mM MgC1z, and every 1.5 mM] nucleotide, and when low temperature of 37 degree [between amplification] C is raised to 45 degrees C~58 degreeC, much more good singularity and efficiency of amplification are acquired. It was found out that DMSO does not need because of amplification or it is not desirable. ■ Homozygosity deficiency of Mo1t4 cellular in to a GM2064Mo1t4 cellular in delta- Pimp bottle field is represented for amplification of a 119 base-pair fragment on a 1 human beta-globin gene, and it obtains from a human Kenomic mutant cell Devonge tree, a cam ten, and NJ -- having -- 50mM MCI and 25 mM Tris-HCj! (pH 8) 10mM MgC1t, 200 Bas / - gelatin, 5-M2-mercaptoethanol, dATP, dCTP, TTP, and dGTP of every 1.51M, And it amplified in reaction capacity of 100J11 containing the following primer m:5'-CTTCTGcagCAACTGTGTTCACTAGC-3' (GHlB) 5'-CACaAgCTTCATCCACGTTCACC-3' (G) 119 per house. A small letter shows a mismatch from wild nature type arrangement for forming a restriction enzyme part during arrangement of the above-mentioned probe. To Negative chain , GHlB is an oligonucleotide of complementary 26-base, and contains an internal Pstr part. To one chain, Glf19 is complementary 29-base oligonucleotide, and contains an internal Hind■ recognition site. These primers were chosen by screening a field of a homologous gene first to EURO and a 1 Breadth dIIII restriction site. Next, it is a primer as it indicated for Example 1! The product made from Pl was carried out. The above-mentioned reaction mixture was heated for 10 minutes in 95 degreeC, and polymerase a total of four p1 which was cooled to room temperature next and which was indicated to 1 one example was added to each reaction mixture, and then mineral oil was stratified into each mixture. This reaction mixture was given to amplification of 30 cycles by the next program. It heats from 37degreeC to 98 degreeC over 2 minutes, cools from 98degreeC to 37 degreeC over; 30 minutes, and is immersed in; and 37 degreeC for 2 minutes. After the last cycle, a reaction mixture was incubated for 20 minutes in 65 degreeC, and the last extension was completed. Chloroform extracted mineral oil and it stored a mixture in 1 20 *C. Generally it is amplification output of a total of 10microl in M13mplO cloning vector 0.5n in which a help is possible! 50-m1I Nace and 10mM Tris-HC11 (pH 7, 8) It digested for 90 minutes in 37 degreeC in capacity containing PstI of 10mMMgC4□ and 20 units, and HindI [I of 26 units of 50 m. -A reaction was stopped by freezing in 20 degreeC. TEi Rinse adjusted capacity to 1101t!, and it loaded 100I on a Biogel P-4 spin dialysis column of 1a/. One 0.1a / drawings were collected, and ethanol precipitation is carried out. (It was found out that beta-grotesque bottle amplification output exists all over 2064 samples of GM at this time.) The next experiment pursued a pollution source of primer G118 or GH19. Since other sources of a primer were not obtained, it understood that some clone-ized arrangement probably originated in pollution DNA in a primer, and continued an experiment. An ethanol pellet is re-suspended in water of 15t1!, Next, 50 mM Tris-HCI (pi (7, 8), 10mM Mg (it adjusted to 20delta capacity containing J z, 0, 5 mM ATP, 1101II dithiothreitol, and 400 unit paste gauze.)) [1 A unit is the quantity of enzyme required to bring about 50% of connection of lambdaDNA digested by Hind m in 30 minutes in 16 degreeC in 204 in 5' end concentration 0.12mM (about 300 m/i). This mixture was incubated in 16 degreeC for 3 hours. The 10-d ligation mixture containing Mo1t 4 DNA was transformed into a competent cell of available to -M E, and Cori JM103. Methods for preparing a transformed stock are Messing J and (1981) Ding h5rdC1 eveland S m osium on Macromoleculas:Recom-binant DNA, It is indicated to A, Waltonlii, Else beer, Amsterdam, and 143-163. A total of 651 colorless plaques (and zero blue plaque) were obtained. Among these, 119 pieces (18%) had (+) Insertion part , and 19 pieces (a %) had (-) chain insert portion. If absorbed, they will be most 20 times as many percents of a beta-grotesque bottle positivity plaque in a primer positivity plaque from amplification technique using E and Henow fragmentation of collie polymerase I. In a method of using Klenow fragmentation, it reacted for 2 minutes in 25 degreeC, and next, a heating stage to 100 degreeC was performed for 2 minutes, it cooled, and Klenow fragmentation was added, and a reaction was repeated 9 times. These results are checking singularity by which an amplification reaction which uses thermostable enzyme of this invention was improved. In a cloning experiment after using GM2064 and a polluted primer, 43 pieces (e %) are (+) 1 among 510 colorless plaques! It had an insert portion. This suggests that an abbreviation half of 119 clones from Mo1t4 contains pollution arrangement. Sequence determination of ten of (+) chain clone from Mo1t4 was carried out. Five pieces are normal wild nature type arrangement, and had the variation from C to one T in the third position of the second codon of a gene (cAC->CAT). Sequence determination of four of a pollution clone from GM2064 was carried out, and all four pieces were normal. Using the above-mentioned technique, it can be used in order to amplify and clone-ize a human N-ras carcinogenic gene using a restriction site modification primer, to carry out sequence determination partially and to clone-ize a base pair segment of HLADQ-alpha, DQ-beta, and a DR-beta gene. A specific DNA arrangement probe for an identification Taq ool gene of a DNA arrangement probe for unfilled space and example " Work "7j Jiyuta A and a Taq polymerase gene was obtained by immunological Screwing of a lambdagtl1 expression library below. Full digestion of the DNA of T and the Aqua typhoid fever is carried out by Human beings I, and he is EcoRI 12-Maher Linka (cCGGAATTCCGG). It connected with New England bio-lapse, digested by EcoRI, and connected with lambdagtll DNA ( Blogma by Ochiku ) by which -E-coRI-digestion was carried out and dephosphorization was carried out. Connected DNA was packed and Jigahafuku plus, Strategyen , E, and Cori were transfected at 1 12 stocks Y1090 (it receives from R and Young). CYaung+R, A which screened a library of the beginning of a 2X10' plaque using 1:2000i Shit of rabbit polyclonal antiserum to refined Taq polymerase (refer to Example 1 and Example 6), And R, 11, and Davis (1983) 5cience. 222 It re-screened until it carried out the re-plate of Near 7B-7821 and the candidate plaque by marginal dilution and became uniform (about 3 cycles). It did not react to preimmune serum but refined phage from a candidate plaque reacted to an immune serum. 1 12 stocks Y1089 (R, Young) were lysogenized to E and Cori using candidate phage. Lysogenic bacteria were screened about production of size from IPTG inductivity inductivity fusion Fluorescence -galactosidase reacted to a Taq polymerase antiserum. Affinity refining of the epitope specific antibody was carried out from all the polyclonal antibodies using fusion firefly white matter to which size fractionation of the solid phase was carried out (102 : J, such as Goldstein, L, S, and B (1986), Ce11. Biol, 2076-2087). An antibody by which epitope ["it was lifted"] selection was made was used in western analysis, and lambdagtll phage candidate who is coding NA arrangement in a unique target to Taq polymerase was identified. lambdagt: One lambdagtll phage candidate who calls one chose specifically from all the rabbit polyclonal Taq polymerase antiserums an antibody which reacts specifically with both for rough extraction drawing containing Taq polymerase and Tag polymerase which were refined. This phage /?gt:]

It was used and further inquired. The label of the A rat RI~conformity people Dad fragment of about 115 bp (s) of Thermes Aquaticus DNA was carried out, and To Maniatis etc. formed a Taq polymerase specific probe by the above-shown. In Southern analysis, this probe was used in order to screen T and an Aqua typhoid fever DNA random genomic library. Development of B and a Thermes Aqua Chicks - random genomic library, and screening lambda furzy Sharon 35 (Wilhe1 mine+A) Via the adhesion end, M etc. carried out Annealing Perilla frutescens (L.) Britton var. crispa (Thunb.) Decne. of the above-shown, and connected it, full digestion was carried out by Mandarin and calcium acetate density gradient super-centrifugality (above-shown, such as Mani-atis) refined an arm by which anil was carried out from stuffer (stuffer) fragmentation. Partial decomposition of the DNA of T and the Aqua typhoid fever was carried out by S au3A, and shake sirloin density gradient super-centrifugality refined halving of 15~2 *kb size. A random genomic library was developed by connecting target DNA fragmentation and vector DNA fragmentation by a molar ratio of 1= 1. carrying out the pan cage of the DNA -- and E and Cori -- 1 12-share LE392 -- or it was alike and transfected 802. A library of phage of the 20 containing 99% or more of Recombination object and 000 or more beginnings was amplified on 1 12-share LE392 to E and Cori. lambdagttt: A C135 Taq genome phage library was screened by an EcoRI insert portion to which the radioactive label of one was carried out (above-shown, such as Maniatis). A candidate phage plaque hybridized specifically was refined, and it further analyzed. CH352: One phage called 4-2 emitted four or more T and Aqua typhoid fever DNA fragmentation after digestion by HindI [l (about 8.0, 4.5, 0.8, and 0.58 kb). Four kinds of Hind nlT and Aqua typhoid fever DNA fragmentation are connected with plasmid BSM13" (a, 2kb, vector cloning Systems , San Diego) digested by HindI [[, and it is E, respectively. It clone-ized to Cori at transformation of 1 12-share DG98. C) 135 : : j!i detaches And 1ndI [DNA fragmentation of 4-2 to about 8.0 kb in plasmid pFC82 (11, 2kb), Another side CH35: 4.5 kb And 1ndl' [DNA fragmentation from :4-2 was isolated in plasmid pFC83 (7, 7kb). Polypeptide of a molecular weight of about 60 new kd (s) immunologically relevant to [further] TaqDNA polymerase in this cell it was indicated to be that E containing pFC82 and 98 shares of Cori DG contained heat stability high temperature DNA polymerase activity (the 1st table) is compounded. 8. '7' aq polymerase code field of 0kbHind mDNA fragmentation -- further -- Work day -promotor proximal 2.8kbH1ncl 11 of 8.0kdHind m fragmentation - oral evidence -- it positioned in 718 portion. The subcloning of this field was carried out, and plasmid pFC85 (6, 0kb) was obtained. After incubating with IPTG, E and Cori DG98I [1 A cow containing plasmid pFc85 compound heat stability 7aq polymerase related activity up to 100 times compared with a parent clone (pFC82/DG98) of a basis (the 1st table). A cell containing pFC85 is Taq P although the heat stable DNA polymerase activity of significant quantity is compounded!! A part of 2LDMA arrangement is translated, and accumulation of Taq polymerase related polypeptide of about 60 kd (s) takes place. A star - top-lE and 13/of revelation BS gate DGof heat stable DNA polymerase activity in Cori (#) 98- 643.8 (#) cell is made to increase to a second-half logarithmic phase (+/-IPTG). -0.02pFC82/DG98 2.2 2.7pFc85/DG98 11.9Sound wave processing was Harvest (ed) and carried out, in 75 degreeC, it heated for 20 minutes, and centrifugality of the 10 d was carried out, and transparent top Qing dynasty was measured about DNA polymerase activity in 70 degreeC. (Book) It is introduction of 1 nM dCTP in [1 unit] 30 minutes. A gene of thermostable enzyme of this invention can be made to reveal in various bacteria expression vectors (ATCC39588), for example, DG141, and pPL N mus ATG. Are a derivative of pBl?322 and it connects with a tryptophan promotor operator and an ATG initiating codon so that an operation is possible, or both of these host vectors are Bosome binding sites (DG141), Or it has gene N binding site (+) PL N **5ATG connected with arrangement and an ATG initiating codon including lambdaPt promotor so that an operation was possible. Restriction enzyme processing of either of these host vectors can be carried out by 5acr, and it can flush-end-ize by Klenow or S1 nuclease, and can make a convenient part for inserting a Taq polymerase gene later. A Taq polymerase gene of perfect length was developed from DNA insertion fragmentation by which subcloning was carried out as follows to plasmid pFC83 and rhoFC85. Vector BSM13" (vector cloning Systems , San Diego) it is marketed from CA -- Listen -- digesting in a unique H4nd m partrestore by Kleno- and dNTP and alike using T4 DNA ligase -- ■ Octa nucleotide Tidribka - 5' -- it connected with -CAGATCTG-3' and a plasmid was isolated from the a As+p"1 A time 2 piece degree transformant transformed into E and 98 shares of Cori DG. One of the clones was digested by III and oral evidence 718, and gel electrophoresis refined large vector fragmentation. Next, plasmid pPc83 was digested by III and Hind m, and fragmentation of about 750 bp (s) was isolated. Plasmid pFC85 is digested by H4ndI [l and Human beings 718, and fragmentation of about 2.8 kb (s) is isolated, And it is A time LLII-H side dI [of pFC83 to about 750 bp (s) ] [by three-piece connection! It connected with fragmentation and And dII - people Breadth 118 vector fragmentation of 85M13". E and 98 shares (ATCC A disease 39.768 of deposition on July 13, 1984) of Cori DG were transformed using this connection mixture, and an Amp" colony was chosen after this, and a plasmid (pLsG+) of about 6.75 kb (s) was isolated. Isopropyl beta-D- Thiogalactoside (IPTG) -guidance DG98 cell which Inclusion pLsG+, With natural enzyme isolated from T and the Aqua typhoid fever, plasmid pLsG1 can be used for zero-order [which compounded Taq DNA polymerase which differs in size], and a single ti D N A mold can be formed in accordance with a method recommended by vector cloning Systems . Next, oligonucleotide instruction variation indecement [Zol Ier and S+ith, A 5 Fallen I restriction site can be introduced as a portion of an ATG initiating codon using Nuc, Ac1ds Res, and (1982) 10:6487-6500 (in upper stream of an internal Hind■ part in a coding sequence of a Taq polymerase gene). After finishing about zero copy which similarly can introduce at least the back (from assistant 718 parts to about 0.7 kb upper stream) BPI11 copy of a carboxy terminus of a gene, and can make easy subcloning of a Taq polymerase gene to an expression vector of specific variation guidance, A gene is isolated from BS113" on Yin-tsu - A time stE■ restriction fragmentation of about 3.2 kb (s), It processes by Klenow fragmentation and all four kinds of dNTP (s), And using T4DNA ligase (flush end conditions), it can digest by 5acl beforehand, can restore by Klenow and dNTP, and can insert in the expression vector which processed by cow intestines phospha Tarta and formed a dephosphorization flush end. A transformant obtained by transforming E and Cori DG116 using this connection mixture is screened about production of Taq polymerase. Revelation of enzyme can be checked by Western immuno plot analysis and active analysis. And button dI [[of about 2.8 kb (s) of plasmid pFc85 - oral evidence -- a bigger portion than that of a Taq polymerase gene contained in 718 fragmentation, For example, you can make it revealed by connecting with an ATG initiating codon an amino terminal end H4ndI [l restriction site which codes a Taq auction market gene using plasmid pPL N ass ATG. Revelation output of this fusion object brings about polymerase from which an end of about 66 and 000~68.000 Dalton was cut out. Plasmid pFC85 is digested by Hind■ and this specific development can be performed by processing by Klenow fragmentation under existence of dATP, dGTP, and dCTP. By further processing fragmentation which arose by 81 nucleases, a single-stranded extension is removed, and DNA which arose is digested by 11118, and it processes by Klenow fragmentation under existence of four kinds of all dNTP (s). dephosphorization plasmid I] PtN **s8 which digested a collected fragment by 5acl beforehand, and processed it by Klenow fragmentation under existence of dGTP using T4DNA ligase, and formed an ATG flush end -- it can be alike to T and can connect with it. Next, this connection mixture is used and it is E. Cori DG116 is transformed and a transformant is screened about production of Taq polymerase. After all, revelation can be checked by Western immuno plot analysis and active analysis. Base heat stability polymerase follows an example indicated later, and is Thermes Aquaticus (Thermus1u1umicro). It can refine from a bacterial culture thing containing enzyme by which could refine directly from a culture for 4 s, or Recombination production was carried out with a required slight change in manufacture of a crude extract. It is a 60 g [after Harvest (ing) by centrifugal separation] cell! 50 mM Tris-HCj (It was suspended in buffer solution 75y4 containing pH 8, 1>, and 1 mM EDTA.) A cell was Lysis (ed) in 14, 000~16 and 0OOPSI in a French press, and Tris -EDTA of an addition of four capacity (a00-) was added after this. Sound wave processing was fully carried out, having added buffer solution A (it is to 0.5 v/V% about 5 n+H, NP-40, and Tween 20 in beta-mercaptoethanol, respectively), and cooling this solution. Homogeneous yQ murky solution obtained is further diluted with buffer solution A, and it was made for the last capacity to be 7.5~8 times the start cell weight. This is called drawing part I. It is polymerase activity in drawing part I and a top Fine picture part, 0.025 M TAPS-HCl (pH 9, 2, 20degreeC') 0.002MMgClz, 0.05M KCR11mM2-mercaptoethanol, every 0.2 mM dGTP, dATP, TTP, and 0.1 mFdCTP CCr -- "P, 0.05Ci/++cM, and 12.5 g" -- polymerase (10n+M Tris-FICj!) of activated * salmon sperm DNA and 0.01~0.2 unitIt measured in the 50-m mixture containing pH 8 and 50n?K (it dilutes in gelatin to which autoclave of f and 1■/- was carried out, 0.5%NP-40, 0 or 5% Tween 20, and 1mM 2-mercaptoethanol). One unit is equivalent to output of period 10nFI for 30 minutes. "DNA carried out is a natural manufacture thing of DNA after partial hydrolysis was carried out by DNase I until it changes 5% of DNAs to a part for acid soluble drawing. [of ° activation] reacting for 10 minutes in 74 degreeC -- and the next -- 40 m -- the 50n [/ml] person in 2mMEDTA -- it moved into carrier DNA solution 1.0mff1 (0'c). TCA and 2% Bilorin acid sodium were added 20% of the capacity (1, zero R1). A sample was filtered through the Watt Mann GF/C disk after for 15~20 minutes in 0 degreeC, and 5% of cold TCA-1% Bilorin acid chloride fully washed, and then it washed by 95% of cold ethanol, and dried, and calculated. In Beckmann Tl450-Tarr, carried out centrifugality of the drawing part ■, they were collected by 2 degreeC at 35.000 rpm, for 2 hours, and also Qing dynasty was considered as drawing part ■. After determining the minimum quantity (it was found out that these quantity generally constitutes 0.25~0.3% of the last capacity) of Pori Min (Pol+1inch) P required for making active 90~95% precipitate, Taq polymerase activity was made to precipitate by Borimine P (what was adjusted to pH 7 and 5 IOV/V (BRL, guy cell Burg , MD) %, and carried out autoclave). Borimine P of a suitable level was gradually added to drawing part ■, agitating over 15 minutes in 0 degreeC. Centrifugality of this solution was carried out by 13.00Orpm over 20 minutes in Befukuman JA140-Tarr in 0 degreeC. The activity of upper Slide was measured and a pellet was re-suspended to 0.5x buffer solution A of 115 capacity (what was diluted to I:2 with water). This suspension was re-centrifuged and a pellet was re-suspended to buffer solution A of 1/4 capacity (0 and 4M KCj! are contained). This suspension is fully homogenized and it is 1 device Was there to 4 degreeC. Centrifugality of this gay Ginet was carried out as mentioned above, and it collected and also named Qing dynasty drawing part ■. Parts for protein drawing are collected by "precipitation" in the 75% saturation of ammonium sulfate, and are centrifuged, and they are for To 27.00Orpm, 5W270-Tarr, and 30 minutes and a thin film which surfaced! 50 mM Tris-HCj (pH 8) the inside of 1 m?I EDTA -- re-'g -- it was made muddy. P-cell buffer solution which repeats these stages and contains 80mM KCj! for protein suspension (20mMKPO.) It fully dialyzed by pH 7, 5, 0, and 5 a+M EDTA, 5 mMbeta-mercaptoethanol, 5 w/v% glycerol, 0.5 v/v%NP-40, and Tween 20]. A dialysis thing was moved to a centrifugal bottle and protein collected from a sack which is P-celll Town liquid which contains 80mM KCl2 in this, and was rinsed was added. Centrifugality was performed in 20 and 0OOX g, and time was shortened in 15 minutes. They are P-cell buffer solution and 80mM KCj about a pellet which stored top Qing dynasty and remained! It was alike, and washed and extracted more, and carried out re-centrifugality. Next, top Qing dynasty was mixed and it was named drawing part ■. Drawing part ■ was applied to the 2.2X22-cm column of phospho cellulose balance-ized with P-cell buffer solution containing 80++M KCl. This column was washed with the same buffer solution, and protein was eluted using 2.5~3 time capacity of a To column, and a straight line gradient of 80in P-cell buffer solution~400mM KCl. A part for drawing containing DNA polymerase activity (about 0.18~0.20?I KCj!) was pooled, and it condensed 3~4 times on an Amicon churning cell and Y130 film. About this cell, it is K (it is P-ce11 buffer solution which does not contain j!, and rinsed, and, in addition to drawing part concentration \yJ (0, the 15MKCl last capacity adjustment), considered it as drawing part ■.). Drawing part ■ was applied to heparin sepharose CL-6B column (Pharmacia) 5 A balance-ized by P-cell buffer solution and 0.15?I KO2.0. 15M KCI Ehong street liquid (a~4 capacity of column) washed a column, and protein was eluted by a straight line gradient of 0.15in P-ce11 buffer solution~0.6511 KC/!. 1:10 dilution to a diluent which does not contain gelatin for 5O5-PAEG analysis was performed, and 1:20 continuing dilution to a diluent which contains gelatin of 1■/- in order to use it for measurement of enzyme was performed. About a part for active drawing (eluted by about 0.3 M KCjl!), it is about specific on a super coil DNA mold, nonspecific, and nuclease/ Topoisomerase , It measured by detecting change of a molecular weight of super coil plasmid DNA after incubating with superfluous DNA polymerase by electrophoresis. Contamination of exonuclease was detected after an incubation with a little line DNA fragmentation. It was found out among a peak drawing part that about 88 kd protein is a main band. When this pool was measured for 30 minutes by 55 degreeC by an abbreviation 3~5 polymerase unit / 600ng DNA, and the minimum detection was possible for a part for main drawing called drawing part ■, it had the highest polymerase activity with nuclease activity. Drawing part ■ was dialyzed to 10+nM KPO4 (pH 7, 5), 5 iMbeta-mercaptoethanol, 5% glycerol, 0.2%NP-40, and 0.2% Tween 20 (HA buffer solution). This dialyzed sample was applied to a column of 31nl of a hydroxy apatite, and enzyme was eluted by a straight line gradient of 10in HA Mild town liquid ~250mM KPO= (pH 7, 5). Polymerase activity began to be eluted in 75111M KPO4, was set to 100mM PO, and showed a peak. A part for active peak drawing was measured by 1= 100~1:300 dilution. A dilution thing of 1:10 [as / in a front chromatography stage] in a diluent which does not contain gelatin for 5 DS-PAGE analysis was prepared. In 55 degreeC, it is significant in 5 polymerase unit, halving which does not have nuclease or 2 chain exonuclease activity was pooled, and it was named drawing part ■. Drawing part ■ was dialyzed to solution of 15.2 or 5% of 25mM acetic acid sodium (p) glycerol, 5mMbeta-mercaptoethanol, 0.1 mM EDTA, 0.1%NP-40, and 0.1% Tween 20, and the pH to 5 was adjusted in room temperature. A dialyzed sample was applied to a DEAE-Tris-Acryl-M (LKB) column of 2- balance-ized beforehand, and was washed with the same buffer solution as the next. Halving containing polymerase activity which did not adhere to a column was pooled, and it adjusted to the 50mMNaCin buffer solution1 [same], and obtained drawing part ■. It is the same buffer solution (25mM acetic acid sodium, 50mM Na (J, 5% glycerol, and 0.1 mM EDTA.)) about drawing part ■. It applied to a CM-Tris-Acryl-M (LKB) column of 2- balance-ized by 0.1%NP-40 and 0.1% Tween 20. This column was washed with the buffer solution with same 4~5 capacity of column, and enzyme was eluted by a straight line gradient of 50in acetic acid sodium buffer solution~400mM NaC1. A peak of polymerase activity was eluted in abbreviation 0.15~0.20M NaCj!. Polymerase activity was first diluted with 1:10 in a diluent which does not contain gelatin for 5O5-PAGE analysis, and was measured in 1:300~1:5001 Interpretation . It is DNA polymerase assay salt (25mM TAPS-HCl2.) at 74 degreeC. PI (9, 4, 2, 0mMHgC1z, and 5QmFIKCA) is used, and it is super coil DNA about specific, nonspecific, and nuclease/ Topoisomerase ! Measurement covering an active peak which receives a congratulation type is performed, Furthermore, measurement of nuclease to M13ssDNA and pBI13227 lug Ment was also performed. A part for active drawing which does not contain detectable nuclease is pooled, and it is migration I got it about a silver stain 5 OS-PAGE mini gel top. this result -- a ratio of about 250.000 units / ■ -- a single band of about 88 kd (s) which have activity is shown. It was found out that Taq polymerase refined as indicated for uni-example 6 is not polluted by Taq, nuclease activity, and Taq exonuclease activity. Taq polymerase is preferably stored into storage buffer solution containing each nonionic detergent abbreviation 0.1~about Q used and 5 v/v%. Storage buffer solution is 50 v/v% glycerol and 1100nc KCI preferably, 20 mM Tris-H (J (pH 8, 0) and 0.1 mM Ethyresin amine 4# acid (EDTA)) It consists of LmFI dithiothreitol, 0, 5 v / v%NP-40, 0, and 5v / v% Tween 20, and 20 n/rn1 gelatin, and preferably is stored by 1 20-degreeC. About this stored Taq polymerase, it is 25mMTris -H (it diluted to buffer solution which comprises J (pH 8, 0), 20mM KCl, 1mMbeta-mercaptoethanol, 0.5%NP-40, 0 or 5% Tween 20, and 500i/-gelatin.). Next, 50mM KCI and 10mM TrisHCl (pH 8, 3), 1.5mM MgC1t, 0.01W/V% gelatin, every 200 greetings dNTP, Reaction buffer which contains Taq polymerase / 1 measurement of an every 1 m primer (a target sequence of 500 base pairs on a contrast mold from bacteriophage lambda is defined) and 2.0~2.5 unit in the last capacity of 1004 was prepared. A mold was added to this reaction buffer, and a sample was put into 0.5 and a polypropylene tube of d, and a sample was covered with heavy white mineral oil of 100microl, and evaporation was prevented. When using a contrast mold (bacteriophage lambdaDNA) of lng a target sequence occupied about 1% of the amount of starts of DNA and used the following conditions, at least 105 times as many amplification was attained. A mold mixture was first denatured over 1 minute for 30 seconds in 94 degreeC by placing a tube into Hot bath . Next, a tube was put into Hot bath of 72 degreeC for 3 minutes, and then it put into zero-order [which put a tube into Hot bath of 37 degreeC for 2 minutes] for 1 minute at Hot bath of 94 degreeC. This sample was repeated a total of 25 cycles. In the end of the 25th cycle, a heat denaturation stage in 94 degreeC was omitted, and an incubation stage of 72 degreeC was instead extended for 3 more minutes. After suspending measurement, a sample was cooled radiationally to room temperature, and as it indicated, it analyzed in an example of precedence. A mold can be amplified the optimal using dNTP of different concentration, and different Taq polymerase of concentration. Size of a target sequence in a DNA sample will affect it at the shortest time required for suitable extension (incubation stage in 72 degreeC). In order to acquire the greatest efficiency, a temperature cycle profile should be optimized about each mold which should be amplified. As an example which precedes Taq polymerase which one Penetration refined to it as was indicated to 1 was indicated, it tablet-ized for storage. However, nonionic polymer detergent was not used. As that example was indicated, when activity was measured, it was found out that this enzyme storage mixture is inactivity. When NP-20 and Tween 20 were added to storage buffer solution, sufficient enzyme activity is saved and it was shown that nonionic detergent is required for stability of an enzyme tablet. As the 1-n sample of A story " Yoohit genome DNA some was indicated for Example 5 using Kleno-fragmentation or Taq polymerase of the equivalent number of units, amplification of 20~35 cycles was given, and agarose gel electrophoresis and a sample some analyzed. Primers PCO3 and PCO4 used in these reactions order it composition of a 110bp segment of a human beta-globin gene. Klenow polymerase amplification shows dirt (smear) of DNA observed typically, when using this enzyme, the cause expected -- nonspecific annealing -- and -- essential -- un-- it is extension of a primer to an unrelated genome sequence under stringent hybridization conditions (I X Heno-salt, 37degreeC). Nevertheless, in Southern * Rond , specific 110bpbeta-grotesque bottle target fragmentation was observed on all the lanes. A substantially different electrophoretic pattern in amplification performed using Taq polymerase is seen, and a main band of single What is a 110bp target sequence in this case. This remarkable singularity is based on temperature when a primer is elongated unquestionably. Like [in Kleno-fragmentation amplification], although an annealing stage was performed by 37 degreeC, after making temperature of a Taq-catalyst reaction rise to about 70 degreeC, enzyme showed significant activity. Primer mold by Brid (formed by a7-degreeC) which Matches (ed) poorly was able to dissociate between this varying temperature from 37degreeC to 70 degreeC, and it was able to use highly by a time of a reaction mixture reaching enzyme activity-ized temperature for extension of only a complementary substrate. This singularity brings about a target sequence of a high yield again compared with the same amplification performed using Klenow fragmentation. It is because 11O-Maher's quantity which nonspecific extension output competes effectively about polymerase, and may be made by Klenow fragmentation by this decreases. Courage 1 ts Mo1t4DNA, 50mM KCj!10 n+M Tris (pH 8, 3) and 10+l1M MgCl z, 0.01% gelatin, The every 1 Rhinoceros primer as follows (150bp field) A thing for amplifying: Taq polymerase of 5'-CATGCCTCTTTGCACCATTC-3' (R379), dNTP of every G-3' (R580) 1.5 mM of 5'-TGGTAGCTGGATTGTAGCT (s), and 5.0 UNY / A A sample contained in reaction capacity light of 1004 was amplified. A sample of three additions containing Taq polymerase of 2.5, 1.3, or 0.6 units was prepared. Amplification of 30 cycles was performed by an above temperature cycle in a plane using the following cycle. It heats from 70degreeC to 98 *C over 1 minute, holds for 1 minute in;98-degreeC, cools to 35degreeC245degreeC from 98degreeC, or 55 degreeC over; 1 minute, and is;35degreeC, It holds for 1 minute to 45degreeC or 55 degreeC, and it heats over; 1 minute from 35degreeC, 45degreeC, or 55degreeC to 70 degreeC, and holds for 30 seconds to; and 70 degreeC. At annealing temperature of 35 degreeC, a Taq enzyme dilution thing of 2.5 units / 100t1! gives the best signal:noise ratio in agarose gel electrophoresis compared with other Taq polymerase concentration of all the. In 45 degreeC, Taq enzyme of 5 units / 100I gives the best signal:noise ratio compared with other concentration. In 55 degreeC, Taq enzyme of 11 gives 5 Uniso /100t of best signal:noise ratios and improved yields compared with other concentration and annealing in 45 degreeC. In 55 *C, as for 7'aq polymerase, singularity is quantity (and - Favorite yield is given.) much more. In another experiment, it is human Zenete ink mutant cell Debossary (Human Genetic Mu nucleic acid ntCe1l Depository) about Mo1t 4 DNA, It is every 10 times to cellular in GM2064DNA containing beta- or T-grotesque bottle arrangement obtained from a cam ten and a new chassis, It diluted with various concentration representing a difference of a copy per cell, and amplified by [as having indicated these samples in this example at annealing temperature of 35degreeC and 55 degreeC]. In 35 degreeC, the best thing which can be seen by agarose gel electrophoresis was 1 in 50 cells copy. In 55 degreeC, the best thing seen is 1/5, 000 cell (it compares with low temperature and they are 100 times as many improvement), and it was shown for the singularity of 'l'aq polymerase under these conditions that annealing temperature which rose is important. In the third experiment, DNA from cellular in 368H containing HIV-positivity DNA obtained from New York, B of Silacs , and a Po1esz state university is made the same, To DNA from SCI cellular in (beta-cellular in which is homozygosity about; sickle cell allele deposited with ATCC on March 19, 1985, and lacks all HIV arrangement and which was EBV (ed) - transformed), It dilutes with various concentration representing a difference of a copy per cell, andA 115bpeJf region of H1■ arrangementPrimer 5K38 and 5K39 to amplify : AT-3' (SK38) and 5'-TTTGGTCCTTGTCTTATGTCCAGAATGC-3' (SK39) are used to 5'-ATAATCCACCTATCCCAGTAGGAGA, At annealing temperature of 35degreeC and 55 degreeC, it amplified by [as having indicated for this example]. Although only undiluted 368H sample was detected at annealing temperature of 35 degreeC according to the place which a result depended on agarose gel electrophoresis shows, According to annealing temperature of 55 degreeC, at least ten to 2 dilution could be detected, and 100 times as many improvement in detection was given. It is 1501K (J!) about rabbit reticulocyte mRNA of A story "" Yo In (the Bethesda research Laborado lease) to cDNA. 50 mM Tris-H (J (pH 8, 3), 10mM MgCj!) z, 51M DTT, 0.5mM dATP, and 0.5mM dCTP, 0.5mMTTP, 0.5oM dGTP, and 0.2 n oligo (dT) 12-18 ( Falconia ), It made in reaction capacity of 100J1! containing AMV reverse transcriptase of 40 unit RNasin and (tic [the Promega biotechnology]) five units, and incubated for 30 minutes in 42 degreeC. A reaction was stopped by heating for 10 minutes in 95 degreeC. RNase (solution 24 of underwater 2■/-) of 2 Bas was added to a sample, and it incubated for 10 minutes in 37 degreeC. Kleno-fragmentation performed three amplification reactions using a pair of different primer. Primers PCO3/PCO4 define output of 110bp. R545-/oligo (dT) versus primer 1 25-30 defines output of about 370 bp (s), and is 1 pair of primer PCO3/oligo (dT). 25-30 defines output of about 600 bp (s). To a human beta-globin gene, PCO3 *PCO4 and R545 are complementary, and have two mismatches between rabbit genes. PCO3 and PCO4 are indicated for Example 1. R345 -- arrangement: -- 5' -- it has -CAAGAAGGTGCTAGGTGCC-3'. Amplification reactions are 50nM NafJ and 10mM Tris41 (J!). (p) 17, 6, 10mM MgCj!2.200 Nitrogen / - gelatin, and 10%DMSO11 voice PC03, I?S45.1 Oh PCO4, or oligo (4T) 25-30.1. 5 mM dATP, 1.5 It carried out using 1/20 of above cDNA (s) (5 m) in reaction capacity of 100I containing mM dCTP, 1, 5 nM TTP, and 1.5 mM dGTP. A sample was heated for 5 minutes in 98 degreeC, and then it cooled to room temperature, and stratified mineral oil of 100IJ1. This sample was given to amplification of 10 cycles using the next program, using machinery indicated for Example 1.1) It was sufficient in 2 or 5 minutes in a heating block, heated from 37degreeC to 98 degreeC, and cooled from 98degreeC to 37 degreeC over [ Degeneration ;2] 3.0 minutes, a Klenow fragment of Annealing :31 unit was added, and it held for 20 minutes in; and '4 37-degreeC (extension). Electrophoresis on 2% agarose gel analyzed 1/20 of each samples (7J). After an ethidium bromide dyed, a different band in 4 samples of PCO3/PCO (s) and an R545-/oligo (dT) sample was seen. Size of a band was in agreement with prospective length. That is, about the former, it was 110bp and they were about 370 bp (s) about the latter. There was no proof of amplification of about 600 bp fragmentation by one pair of PCO3/oligo (aT) primer. Southern blotting of the content of gel is carried out to Genan tolan (Genan tran) nylon membrane, And you made it hybridize with human beta grotesque bottle probe pBR328:betaA which is indicated to the 5cience % above-shown, such as 5aiki, by a standard method and by which nick translation was carried out. Obtained auto radio Durham extended a conclusion which reached before. That is, fragmentation of 110bp and about 370 bp (s) is beta-grotesque bottle specific amplification output, and significant amplification of about 600 bp band was not detected. A sample of three additions was amplified by Taq polymerase produced by performing it above using one pair of same primer as the above. They are 54 portions of cDNA 50mM MCI and 25 mM Tris-H (1/!) (pH 8, 0) 10 mM?Ig (f !, 200 codfishes / - gelatin) 10% Leaves 5O21 team PCO3 or R545, one voice PCO4, or oligo (dT) It amplified in reaction capacity of 100I containing 25-30.1, 5mM dATP, 1.5d dCTP, 1.5-ml TTP, 1, and 5 IIM dGTP. A sample was heated for 5 minutes in 98 degreeC, and it cooled to room temperature. 1! Taq polymerase (1 / 8 dilution thing of loft 2) of J1 was added to each, and mineral oil of about 100 IJ (s) 1 was stratified. this sample was indicated above using the next program -- amplification of 9 cycles was very given in a device of Teal (Peltier). 1) It heats from 35degreeC to 60 degreeC over 1 minute, heats from 60degreeC to 75 degreeC over [;2] 12 minutes, heat from 70degreeC to 95 degreeC over [ Elongation ;3] 1 minute, and it is immersed in Degeneration :4 95-degreeC for 30 seconds, and cools from 95degreeC to 35 degreeC over [;5] 1 minute. ( Annealing ) It was immersed for 30 seconds in; and 6 35-degreeC. The last (10th cycle) extension was completed by incubating a sample in 70 degreeduring 10 more minutes C after the last cycle. Each last capacity was about 100 Jd (s). Like the above, 1/20 of each sample (10td) were analyzed on 2% agarose gel. In this gel, amplification output existed in all three samples. That is, about about 370 bp (s) and PCO3/oligo (dT), they were [4 / PCO3// PCO / oligo / R545// 110bp and / (dT) ] about 6 oobp (s). These results were checked by hybridization with Southern shift and a pBR328:betaA probe. Generation of 600bp output of Taq polymerase suggests that Taq polymerase can make DNA still longer than Klenow fragmentation generate intentionally [are not by Klenow fragmentation and]. A following bacteriophage and a bacteria stock are Citadel master culture collections (ce tusMaster Cu1ture CollCo11ection) (c). It Deposit ed in the U. S. power Liaonia , Emeryville, 1400.53 street, an American type culture collection (ATCC), the U. S. Moray land, Rockville, and the 12301 park round live. These deposition was performed on a basis of regulation of a rule (Budapest Treaty) revealed to Budapest Treaty about international recognition of deposition of a microorganism for patent procedure, and it. This guarantees maintenance of a survival culture ranging from a day to 30 years of deposition. These microorganisms will be made available by ATCC at a basis of regulation of Budapest Treaty, and will be made available in accordance with a contract of this applicant and ATCC which guarantee availability which is not limited after issue of a related United States patent. Don't understand it as the availability of a deposited microorganism being consent of carrying out this invention against a right observed in a basis of authority of one of the governments in accordance with that Patent Law. CH35:Taq#4-2 3125 40.336 May 29, 1987 E -- if it summarizes on Cori DG98/pFC May29, 4211987 [83312867 or] -- this invention, A method for amplifying 1 or a plurality of specific nucleic acid arrangement using a temperature cycle chain reaction and thermostable enzyme is provided, and reaction primer extension output which may function as a useless mold of the next primer extension reaction in this method is generated. This method is useful, when detecting nucleic acid arrangement which accepts a small quantity very much first and exists, and especially when detecting nucleotide change using an arrangement specific oligonucleotide. A method of this invention provides stages fewer than indicated [required to perform an increase in a yield of amplified output, higher singularity, and an amplification method] conventionally.

[Brief Description of the Drawings]

Drawing 1 is a restriction map of plasmid pFC83 containing the Hind m 1. Aquatics DNA insert portion of about 4.5 kb by which subcloning was carried out into plasmid 85M13". Drawing 2 is a restriction map of plasmid pFC85 containing And ind m -Asp718 T of about 2.8 kb by which subcloning was carried out into plasmid BSM13", and a Aquatics DNA insert portion.