A method of DNA amplification.

0001This invention relates to a method of DNA amplification. In particular, the invention relates to an improved PCR method of DNA amplification which is useful in a wide range of medical and bio-engineering fields such as the construction of DNA libraries of specific chromosome regions, the isolation of genes originating in specified chromosome regions and probe development.

0002In recent years, rapid progress has been made with molecular biological elucidation of the structures and mechanisms which constitute life and with the accompanying technical applications, and the foundations of genetic engineering and biotechnology have gradually been established.

0003Using such techniques, various genes which are characteristic of species and individual structures have been identified extending from procaryotes to eucaryotes and much knowledge has been amassed by the analysis of this genetic information. This research is now being extended to the genomic DNA of higher animals, including man.

0004In order to determine the base sequence of the total human genome, which is said to be composed of 3,000,000,000 base pairs, and to draw up its genetic map, the construction of DNA libraries for specific chromosome regions and the drawing up of physical maps of the chromosomes using individual DNA fragments as indicators (probes) has become an essential process. Furthermore, the physical maps of such chromosome specific regions can be an effective means of identifying the gene responsible for most human genetic disease which has not as yet been elucidated at the molecular level and for analyzing its structure.

0005In the past, a chromosome DNA library has been built up by cloning fragmented chromosomal DNA in a cloning vector, and collecting individual clones to form a library. In this way, in order to study the human genome, specific chromosome libraries constructed from sorted chromosomes or hybrid cells have been used, and, jumping libraries and yeast artificial chromosome (YAC) libraries and the like have been prepared and used in the development of new probes for investigating the target gene.

0006However, in cases where such conventional gene libraries have been used it takes time to obtain the large number of necessary probes for the specific region of the chromosome and, as a result, the need for much effort, time and expense for the elucidation of the target gene cannot be avoided.

0007On the other hand, methods in which DNA libraries and probes have been prepared by amplifying DNA fragments of physically cut-out chromosome specific regions using the PCR (polymerase chain reaction) method have been suggested in recent years (for example, Ludecke et al., Nature, <u>338</u>, 384, 1989). The PCR method involves the use of synthetic oligonucleotides complementary to a certain base sequence of a template DNA as primers for <u>in vitro</u> DNA synthesis to enable DNA fragments which are more or less the same as the template DNA to be obtained in large amounts by repeating a cycle of annealing, extension and denaturation. Automation of this amplification process has lead to its application to gene cloning.

0008However, use of the PCR method requires prior knowledge of the base sequence at least at both ends of the template DNA fragment. Thus in cases where the method is applied to DNA fragments of unknown sequence derived from chromosome specific regions, ligation of the individual DNA fragments with vectors etc. is essential and the template DNA is accordingly subjected to various chemical and enzymic pre-treatments. Consequently, ligation PCR of this type is not only a more complicated operational procedure than the PCR method used for known DNA sequences but the amplification efficiency is also reduced and there is a disadvantage in that many DNA fragments are required for templates for example.

0009The present invention provides a new method of DNA amplification in which trace quantities of DNA fragments of unknown sequence can be amplified simply and efficiently.

0010This present invention provides a method of amplifying template DNA by the PCR reaction wherein (a) a single oligonucleotide is contacted with said template DNA whereby said oligonucleotide randomly anneals to a single strand of said template DNA and DNA sequences complementary to the said single strand are synthesized using said oligonucleotide as a primer and said oligonucleotide also randomly anneals to the complementary strands whereby DNA sequences corresponding to the template DNA are synthesised so that PCR amplification yields synthetic DNA sequences which each incorporate said oligonucleotide sequence at the 5'end and the complement of said oligonucleotide at the 3'end, and (b) said synthesised DNA sequences then being subjected to PCR amplification using said oligonucleotide as a primer.

0011Viewed from another aspect, the present invention provides a gene library comprising cloned sequences amplified by means of the above described method.

0012Furthermore, in a preferred embodiment of the DNA amplification method of the present invention the template DNA and oligonucleotide are annealed for 90 - 150 minutes in a reaction solution at 10 - 40°C which contains an amphipathic polymer such as polyethylene glycol etc.

0013Figure 1 is an agarose electrophoresis pattern (A) of DNA fragments amplified by the method of this present invention and a Southern blot pattern (B) in which human genome total DNA had been used as a probe.

0014Figure 2 is an agarose electrophoresis pattern which illustrates the insert size of the λphage clone in which the DNA fragment amplified by the method of this present invention has been introduced.

0015Figure 3 is an agarose electrophoresis pattern of DNA fragments tested with the method of this present invention.

0016In the present invention, the DNA sequence which is the subject of the amplification is a DNA fragment for which base sequence information has not been obtained (at least, a DNA fragment for which the base sequence information required for conventional PCR amplification has not been obtained), such as a chromosome specific region which has been physically cut out or a DNA sequence which has been cloned for a YAC library etc. for example. In the past, the ligation PCR method as described earlier could be used in cases where such DNA fragments were PCR amplified, but the method of this present invention is such that DNA fragments of unknown sequence can be amplified very effectively using more or less the same procedure and apparatus as used for conventional PCR without carrying out a chemical or enzymatic pre-treatment of the template DNA like that in the case of the ligation PCR method.

0017The method of the present invention involves first annealing a single type of oligonucleotide in an arbitrary region of a strand of the template DNA which has been thermally denatured, then extending the DNA chains which are complementary to the template DNA using this oligonucleotide as a PCR primer, and synthesising the complementary DNA corresponding to a random sequence region of the template DNA. Thus, by repeating the cycle of denaturing, annealing and extension, various DNA sequences having the primer sequence at one end and a sequence complementary thereto at the other end thereof corresponding to a random region of the template DNA may be synthesised (this process is referred to hereinafter as low stringency PCR).

0018It will be appreciated that the single oligonucleotide also anneals to the complementary strand to enable PCR amplification to take place.

0019Low stringency PCR differs from ordinary PCR in that it uses as a primer a single oligonucleotide of which the whole sequence is unlikely to be complementary to any sequence of the template DNA. A single primer of 20-30 nucleotides which has a plurality of restriction enzyme recognition sequences and which has at least two bases on the 3'-end which do not form restriction enzyme sites may be used. Thus, since random base sequences exist in the sequence of the DNA chains, the sequence of the 3'-end of the above mentioned nucleotide can provide a PCR primer in any region of the template DNA by annealing to complementary sequences on the template DNA. Furthermore, in cases where the above mentioned oligonucleotides are used as primers, the various DNA sequences which are ultimately amplified have a plurality of restriction enzyme cleavage sites at both ends and ligation to a vector etc. can be carried out easily. The synthetic nucleotide comprised of the base sequence 5'-TAGATCTGATATCTGAATTCCC-OH3' (referred to hereinafter as BVE22cc) can be cited as an actual example of such an oligonucleotide. BVE22cc has within the sequence comprised of 22 nucleotides recognition sequences for the restriction enzymes BglII, EcoRV and EcoRI (AGATCT, GATATC, GAATTC respectively) and it has a continuous sequence of cytosine (C) at the 3'- end. The DNA sequence which is amplified by means of the present invention can be used directly as a probe for the screening of genome libraries for example, and in this case it is also possible to use oligonucleotides which do not include a restriction enzyme recognition sequence.

0020However, as mentioned above, the low stringency PCR of this present invention involves the use of a oligonucleotide of which the complementary sequence for the template DNA is small and so the efficiency of the annealing of the template DNA and the primer is low.

0021Thus, with the method of the present invention it is recommended that the annealing reaction of the template DNA and the oligonucleotide in the low stringency PCR is carried out over a long period of time. That is to say, whereas the annealing reaction in ordinary PCR is of the order of 1 - 2 minutes, in the present invention it is set at 90 - 150 minutes and the opportunity for the template DNA and the primer to approach and hybridise is increased.

0022Furthermore, this annealing reaction is preferably carried out at low temperature. Ultimately, with ordinary PCR, the template DNA and the primer are annealed in a reaction solution at 50 - 60°C, but in such a temperature range the free primer and template DNA in the reaction solution are subject to the effects of thermal vibration and so mutual annealing is difficult even when they approach. Hence, the temperature of the reaction solution during the annealing reaction is set to a low temperature of 10 - 40°C and the effect of thermal vibration is minimised.

0023Moreover, in the method of the present invention an amphipathic polymer such as polyethylene glycol (PEG) or a polysaccharide such as glucose or sucrose for example may be added in order to increase the annealing efficiency of the template DNA and the primer. Ultimately, these amphipathic polymers increase the viscosity of the reaction solution and suitably suppress the brownian motion of the free template DNA and primer and increase the unification potential, providing conditions which are suitable for annealing. Moreover, these amphipathic polymers have no chemical action on the DNA molecule and no effect on the denaturation and extension steps in the PCR.

0024These means of increasing the annealing efficiency in low stringency PCR can be adopted suitably either individually or in various combinations according to the added amount and molecular weight of the oligonucleotide which is used for the primer and/or the template DNA which is the subject of the amplification for example.

0025Next, the various DNA sequences synthesised by the low stringency PCR mentioned above are amplified without changing the sequences by means of PCR under different conditions (referred to hereinafter as high stringency PCR). In this case, the DNA sequence which is the subject of the amplification, as aforementioned, has the primer sequence at one end and its complimentary sequence at the other end and so the oligonucleotide used in the low stringency PCR is used for the primer. Moreover, if the primer is of a single type and a plurality of templates (DNA sequences) is excluded, the high stringency PCR is such that template DNA is amplified according to the number of cycles in accordance with more or less the same general principles as the conventional PCR. Furthermore, the conditions for denaturation, annealing and extension can be set in the same way as for conventional PCR, or the respective conditions can be modified somewhat and carried out in a number of stages.

0026The various DNA sequences amplified in this way may be used as probes for genetic screening for example since they include random sequence regions of the template DNA and they are comprised of the quantities required for the various genetic operations, or they may be cloned into a vector to establish a gene library.

0027Such DNA sequences may be further characterised following amplification by sequencing.

EXAMPLES

0028Examples are described below and the DNA amplification method of the invention is described in practical terms, but of course the invention is not limited to the cases indicated below.

0029Moreover, in the following examples the high stringency PCR is carried out in two stages, changing the conditions, and so the low stringency PCR is indicated as the 1st PCR and the two types of high stringency PCR are indicated as the 2nd PCR and the 3rd PCR.

Example 1

0030Chromosome DNA recovered from two metaphase chromosome 4 50 - 100% fragments (ie. fragments of the distal half of the short arm of human chromosome 4) was synthesised and amplified using the method of the present invention.

0031First of all, for the 1st PCR, 2 µl of the 1st PCR solution was added to a tube which had been filled with 1 µl of pickup solution (composition as indicated in Table 1) which contained chromosome DNA fragments and a cycle of denaturation (90°C, 5 min), annealing (22°C, 120 min) and extension (50°C, 20 min) was repeated six times using a DNA thermal cycler (made by Perkin-Elmer-Cetus Co.). Furthermore, at this time the temperature was raised gradually from 22°C to 50°C over a period of 20 minutes from annealing to extension in each cycle. Moreover, the reaction solution composition used in the 1st PCR was as shown in Table 1.

0032Next, after completing the 1st PCR, 6 µl of the 2nd PCR solution (same composition as in Table 1) was added to the reaction product (3 µl) to make a total of 9 µl, and the 2nd PCR was carried out by repeating the cycle of deneturation (92°C, 1.5 min), annealing (50°C, 1 min) and extension (72°C, 2 min) eleven times. However, on the first cycle only, the denaturation was carried out for 5 minutes at 92°C.

0033Finally, for the 3rd PCR, 51 µl of the 3rd PCR solution was added to the 2nd PCR product (9 µl) to make a total volume of 60 µl, and a cycle of deneturation (92°C, 1.5 min), annealing (55°C, 1 min) and extension (72°C, 2 min) was repeated thirty times. Moreover, the composition of the 3rd PCR solution was as shown in Table 1.

0034This accumulation of DNA chains for which the chromosome DNA amplified by the three stage PCR was the template was analyzed using the agarose electrophoresis method and the Southern blot method using full-length human genomic DNA as a probe. Moreover, as a comparative example for this analysis, amplification with the same method as described above was also carried out with 1pg and 10fg of RsaI digested human genome DNA (positive control) and metaphase chromosome DNA which had been completely sublimed by laser irradiation and with no template (negative control).

0035The results were as shown in the agarose electrophoresis pattern (A) and the Southern blot pattern (B) of Figure 1.

0036Moreover, in Figures 1 (A) and (B), Lane 1 shows the state of amplification of the DNA of metaphase chromosome 4p 50 - 100% fragments, Lane 2 shows the state of amplification of the metaphase chromosome DNA which had been completely sublimated, Lanes 3 and 4 show the states of amplification respectively of the 1pg and 10fg RsaI fragments of human genome DNA, and Lane 5 shows the state of amplification in the case where no template DNA was added. Furthermore, in Figure 1(A), Lane 6 shows the electrophoresis pattern for the PstI fragment of the λ phage DNA (referred to hereinafter as λ/PstI) used as a size standard.

0037As is clear from Figure 1, in the method of the present invention the DNA fragment of unknown sequence cut out from the chromosome specific region (chromosome 4p 50 - 100%; Lane 1) was amplified with good efficiency in the same way as the RsaI fragment of human genome DNA (Lane 3 and 4). Moreover, the average sizes of the respective amplification products were about 500 bp for the human genome DNA fragment, and a smaller size of about 300 - 400 bp for the chromosome DNA fragment, implying that there is some mechanical or chemical damage during chromosome preparation. Furthermore, faint bands were obtained on amplifying the sublimated chromosome (all burned down metaphase; Lane 2) and in the absence of a template (no template; Lane 5), but these were thought to be due to inadequate sublimation treatment (laser irradiation) of the chromosome or to originate from some contamination in the manipulation process for example. In any case, there is a clear difference on comparing the amplification efficiency in the presence of the proper template DNA.

Example 2

0038The PCR products of the chromosome DNA obtained in Example 1 were each cloned in a λ phage vector and a library was built up.

0039First of all, the DNA was extracted from about half of the 3rd PCR solution in Example 1 by extraction with phenol/chloroform and chloroform/isoamyl alcohol, and by ethanol precipitation, and this DNA was treated with EcoRI to create cloning sites on both ends. After removing the very small DNA fragments by ultra-filtration, the fraction of amplified DNA (50 - 100 ng) was introduced and ligated to the EcoRI sites of λ phage gt11 which had been pre-treated with phosphatase and this was packaged <u>in vitro</u> and used to infect E. coli Y1090r cells. As a result 5 x 10⁶ clones were obtained.

0040Next, 68 clones were selected randomly from this λ phage library and the inserts characterised. That is to say, the insert was amplified by means of conventional PCR taking each clone as template DNA and using a primer of the same sequence as the EcoRI part of the λ phage and the sequence adjacent thereto, and the size was analyzed by means of agarose electrophoresis.

0041The agarose electrophoresis patterns for 20 of the 68 types of insert analyzed are shown in Figure 2. The results showed that the insert size of the 68 clones analyzed was distributed over about 100 - 1500 bp, and this is in close agreement with the size distribution of the amplified chromosome DNA in Example 1 (see Figure 1). Furthermore, when these inserts were hybridised with human genome DNA (EcoRI or HindIII fragments), 40 - 60% of the clones were confirmed as being a human unique sequence, about 10% were confirmed as being a high frequency repeat sequence and 20 - 30% were confirmed as being a low - intermediate frequency repeat sequence.

Example 3

0042The optimum concentration range of the primer and/or amphipathic polymer in each PCR was tested.

004310fg of RsaI digested human genome DNA was amplified in the same way as described in Example 1. The 1st PCR was carried out with a PEG concentration of 0, 8 or 12% in the reaction solution and with a primer (BVE22cc) concentration 0.2, 0.5 or 1.0 µM. After completing six PCR series, one sixth of each reaction solution was subjected to electrophoresis on 1.5% agarose, stained with ethidium bromide and the respective reaction efficiencies were analyzed.

0044The results of this experiment were as shown in the agarose electrophoresis patterns (1st PCR) of Figure 3. Moreover, in Figure 3 (1st PCR), Lane 1 shows the λ/PstI used as a size standard, and Lanes 2 - 4 show the states of amplification of the DNA fragment (10fg) with a primer concentration of 1.0 µM and PEG concentrations of 0, 8 and 12% respectively, while Lanes 5 - 7 show the states of amplification of the DNA fragment (10fg) with a PEG concentration of 12% and primer concentrations of 0.2, 0.5 and 1.0 µM respectively. As is clear from Figure 3 (1st PCR), the efficiency of the DNA chain synthesis increased as the PEG concentration was increased when the primer concentration was set at 1.0 µM (Lanes 2 - 4), and the efficiency of the synthesis increased as the primer concentration was increased when the PEG concentration was set at 12% (Lanes 5 - 7). However, although not shown in Figure 3, the synthesis of DNA chains was also observed in cases where no template DNA was present with a PEG concentration of more than 15% or a primer concentration of more than 3 µM. Consequently, it was adjudged that when BVE22cc is used for the primer and PEG is used for the amphipathic polymer in the low stringency PCR of this present invention, a PEG concentration in the reaction solution set within the range 10 - 15% and a primer concentration set within the range 1.0 - 1.5 µM is desirable.

0045Next, the solution which contained 12% PEG and 1.0 µM primer from among the various reaction solutions used in the above mentioned 1st PCR was treated to a 2nd PCR using the same method as described in Example 1 and then it was submitted for a 3rd PCR. The 3rd PCR was carried out with the adjustment of the primer concentration in the reaction solution to 3.0, 4.0 or 6.0 µM.

0046The results obtained were as shown in Figure 3 (3rd PCR).

0047Moreover, in Figure 3 (3rd PCR), Lanes 8 - 10 show the states of amplification when the primer concentration was set at 3.0, 4.0 and 6.0 respectively. As is clear from Figure 3, of the three primer concentrations tested, the amplification efficiency with 6 µM primer was the best. However, DNA chains which had a smaller average size than the DNA chains synthesised at the other primer concentrations (3.0 or 4.0 µM) or in the 1st PCR (PEG 12%, primer 1.0 µM) were amplified at this concentration. Consequently, it was adjudged that in the high stringency PCR a primer concentration set to some 4.0 - 5.0 µM in the reaction solution in the final stage is desirable. <tables id="tabl0001" num="0001"><img file="EP0543484A2_D0001.tif" /></tables>