Improved method for assessing the quantity of nucleic acids reagents combination and a kit a

(54) IMPROVED METHOD FOR DETECTING NUCLEIC ACIDS AND REAGENT COMBINATION AND CUTTING TO ITS IMPLEMENTATION (57) The invention relates to a method for detecting nucleic acids by hybridization, wherein the capture probe used are modified primers incorporated into copies of the nucleic acid to be detected, and then detect the copies by at least one selective detector probe.

Furthermore, the invention relates to a method for the detection of nucleic acids by hybridization, wherein the detector probes used as modified primers, which are incorporated in copies of the nucleic acids to be detected. In this method, the copies of the nucleic acids to be detected in which the modified primers are incorporated are first hybridized with at least one selective capture probe, then the hybrid formed is separated by means of the capture probe and finally the present hybrid is detected.

In addition, the invention relates to reagent combinations and cutlery for carrying out the described method.

AT 395 434

TOR G078MB

The invention relates to a rapid and sensitive method for the detection of nucleic acids by means of hybridization techniques, in which the detector probes are incorporated as modified primers in copies of the nucleic acid to be detected before the hybridization reaction is carried out. In addition, the invention relates to a reagent combination and a kit for carrying out this method.

Furthermore, the invention relates to a method for the detection of nucleic acids by means of

Hybridization techniques in which the capture probes are incorporated as modified primers in copies of the nucleic acids to be detected before the hybridization reaction is performed. The invention further relates to a combination of reagents and a kit for carrying out this method.

In hybridization reactions, a labeled oligo- or polynucleotide, ie the probe molecule, is transferred

Base pairings attached to a nucleic acid to be detected. Various hybridization methods have been used to detect nucleic acids. In direct hybridization methods, the sample is either in solution or attached to a solid support. The nucleic acid to be detected is detected with a labeled probe molecule.

U.S. Patent 4,486,539 describes a sandwich hybridization method. Two separate probe molecules are used in this procedure. One of them is a detector probe. It is labeled and used for detection. The other is a capture probe. It is immobilized on a solid support and serves to separate the nucleic acid to be detected from the reaction mixture.

The hybridization method in solution is described in GB-OS 2,169,403. In this method, two different probe molecules are used, which are in the same solution. The detector probe is marked with a detectable mark. The capture probe is provided with a group having an affinity for another component. After hybridization, the hybrid formed between the capture probe, the nucleic acid to be detected, and the detector probe is separated from the hybridization solution using the other part of the affinity pair

The enzymatically catalyzed DNA polymerization in which the nucleotide sequence of a given

Nucleic acid strand, ie a template, is exactly copied and thereby the complementary strand is synthesized, is well known in the art; see. z. Komberg, "DNA replication", WH Freeinan & Co., San Francisco, pp. 221-225 and 670-679, 1980, and Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. P. 122,1982. This biological multiplication is used in hybridization detection methods, that is, in detection methods in which the microorganism to be detected is cultured, thereby enriching its DNA before carrying out the test; see. z. B. Woo, Methods Enzymol. 68 (1979), p. 389, and U.S. Patent 4,358,535. Specific DNA sequences can also be amplified in living cells, e.g. By using suitable drugs; see. z. Clewell and Helinski, J. Bacteriol. 110 (1972), p. 1135, and EP-A 55 742. More specific DNA enrichment is described in EP-A 175 689. In this case, the nucleic acid to be detected is combined with a plasmid replicon and introduced into a suitable cell. Another method is described in EP-A 201184. The primer-dependent DNA synthesis is used in an in vitro reaction for the amplification of the DNA to be detected. EP-A 200 362 proposes a method for the detection of amplified nucleic acid sequences which, however, is not fast, sensitive, especially for carrying out serial tests and easy enough

Accordingly, it is an object of the invention to provide a rapid and simple method of detecting low levels of nucleic acid.

According to the invention, this object is achieved by providing a hybridization method in which either the detector probes or the capture probes act as modified primers which are incorporated into the copies of the nucleic acid to be detected during a template-dependent polymerization reaction prior to carrying out the hybridization, in which the nucleic acid to be detected then amplifies becomes.

In the method according to the invention at least one primer is needed, which is always modified. If the

Detector probes serve as primers in the polymerization reaction, the primers have at least one suitable, detectable label or at least one specific site to which at least one suitable, detectable label can be bound.

In another embodiment, the capture probes are used as primers in the polymerization reaction. In this case, the primers have at least one suitable part of an affinity pair or at least one suitable site to which at least one suitable part of an affinity pair can be bound.

The invention also provides a reagent combination and kit for carrying out the method of the invention. In a preferred embodiment, the reagent combination is in a subdivided container.

By using the detector or capture probes as primers in a polymerization reaction, it is possible to increase the sensitivity of the hybridization reaction by several orders of magnitude over detection methods in which the nucleic acid to be detected is measured directly. In addition, will

According to the present invention, a convenient method for carrying out the hybridization reaction in solution is provided so that the hybrid molecules can be readily and rapidly separated from the hybridization solution after the hybridization reaction.

The erfmdungsgemäße method is particularly useful for the diagnosis of certain diseases that are difficult to diagnose with conventional methods. Therefore, the method according to the invention is particularly suitable for the identification of cytomegaloviruses and HIV or AIDS viruses. The figures show:

Fig. 1 shows the nucleotide sequence of the modified primers (P_a and P_b) and the selective probe molecules (Si and S2) used in Examples 1 to 3. Further, it shows the relative positions of the modified primers (P_a and P_b) and the selective probe molecules (Sj and S2) in the case to be detected in this case

Nucleic acid. The solid lines (A and B) denote the two strands to be detected which are in both

Directions continue. The arrowheads on the primers (P_a and P_b) indicate the direction in which they are extended during the polymerization.

Fig. 2 shows the nucleotide sequence of the modified primers (P_a and P_b) and the selective probe molecule (S) used in Example 4. Further, it shows the relative positions of the modified primers (P_a and P_b) and the selective probe molecule (S) in the nucleic acid to be detected in this case. The line (A) denotes the RNA and its identical DNA copies, and the line (B) shows the complementary DNA copies. The arrowheads on the primers (P_a and P_b) indicate the direction in which they are extended in the polymerization.

Preparation of probe molecules

The probe molecules used according to the invention are oligo- or polynucleotides. They can be produced synthetically or semi-synthetically, depending on which preparation method is preferred for the probe molecules to be used as primers. In addition, the probe molecules can be processed by recombinant DNA or produced from directly isolated from nature nucleic acids. A probe molecule can be bound to a suitable vector. It may optionally contain vector parts. A series of usable according to the invention

Primer and probe molecules are commercially available.

The detector probes as modified primers

In one of the methods according to the invention, the detector probes are oligo- or polynucleotides which are bound to the nucleic acid to be detected via base pairings and serve as primers for a template-dependent, enzymatic nucleic acid synthesis. It is essential that the primers acting as a detector probe have at least one suitable, detectable label or at least one specific site to which at least one suitable, detectable label can be bound.

Various radioactive isotopes or radioactively labeled compounds can be used as the label. The labeling substance may also fluoresce, luminesce, light-emitently or be enzymatically or immunologically detectable. It is also possible to use labels which are based on the affinity of biotin

Avidin or streptavidin, lanthanide chelates, ferritin and heme compounds. Further examples are immunologically detectable haptens, such as AAF and AIF (Acetoxyacetylfluorenderivate). Identification may also be performed using mediating molecules, e.g. As proteins.

The process according to the invention does not depend on the label used. All currently known

Labeling substances that are suitable for the hybridization of nucleic acids can be used. However, it is essential that the label be selected from a group of labels that do not interfere with the function of the primer, as long as the detector probes are used as primers. The primer used as a detector probe must be provided with the label so that the nucleic acid polymerase still recognizes the primer as such.

The capture probes as modified primers

In another method of the invention, the capture probes are oligo- or polynucleotides which are linked to the nucleic acid to be detected via base pairings and act as primers in a template-dependent enzymatic nucleic acid synthesis. It is essential that the primers acting as a capture probe have at least one suitable part of an affinity pair or at least one specific site to which at least one suitable part of an affinity pair can be bound. The part or parts of the affinity pair can also be bound via a mediating molecule to the primer acting as a capture probe. The only conditions are that the hybrid can be separated from the hybridization solution with the aid of the affinity pair and that the function of the primer is not impaired.

The part of the affinity pair is a constituent with an affinity for another constituent. Examples of such affinity pairs are biotin-avidin or streptavidin, a heavy metal derivative - a thio group, various homopolynucleotides such as poly dG - poly dC, poly dA - poly dT and poly dA - poly U. It is possible

But also use other pairs of compounds, provided that they have sufficient affinity for the specific binding of the modified captive probe primer incorporated into the copies of the nucleic acid to be detected to the solid support. Suitable affinity pairs are also ligands and conjugates used in immunological procedures.

The selective capture probe

In one of the methods of the invention, where the detector probe is used as a primer, a selective capture probe is required to allow for the selective separation of the copies of nucleic acid to be detected into which the modified primers are incorporated. It is essential that the capture probes be sufficiently homologous to the nucleic acid to be detected to allow their specific hybridization with the copies of the nucleic acid to be detected and thereby facilitate the selective separation and detection of the detector probe primers incorporated into the nucleic acid replicas ,

The selective detector probe

In another method of the invention, where the capture probes are used as modified primers, a selective detector probe is required to allow for the detection of copies of the nucleic acids to be detected, into which the modified primers are incorporated. It is essential that the detector probe is sufficiently homologous to the nucleic acid to be detected in order to specifically hybridize with it and thereby to selectively identify the nucleic acid to be detected. The detector probes may have suitable, detectable labels, for example those mentioned above.

reagents combinations

The detector probe as a modified primer

The present invention relates to a reagent combination containing at least one modified

A primer having at least one suitable detectable label or at least one specific site to which at least one suitable detectable label can be attached, and at least one selective capture probe having at least a portion of an affinity pair or at least one specific site to which at least a part of an affinity pair can be bound.

The capture probe as a modified prima

The present invention also relates to a reagent combination containing at least one modified primer, since at least one suitable part of an affinity pair or at least one specific site to which at least a suitable part of an affinity pair can be attached, and at least one selective one

Detector probe having at least one suitable, detectable label or at least one specific site to which at least one suitable, detectable label can be bound.

The invention also describes a kit suitable for the detection of nucleic acid. The cutlery, optionally packaged in a compartmentalized container, contains one of the abovementioned reagent combinations in combination with at least one of the following reactants or the following equipment needed in the test procedure, ie optionally a vessel containing at least one reagent for the template-dependent polymerization, optionally a vessel containing the four deoxynucleoside triphosphates, optionally a device suitable for carrying out the polymerization and hybridization, and suitable means for detecting the nucleic acid to be detected, and optionally a device suitable for detecting the marking. The preferred devices and reactants are explained in more detail below.

In the method of the invention, at least two modified primers are first added to a denatured sample solution. Both primers are primers either as a detector probe or as a capture probe. In each case, the modified primers attach to the complementary strand of the nucleic acid to be detected, ie the template. After the addition of an enzyme that synthesizes template-dependent nucleic acid, the

Primer extension This method is used in vitro with high yields to create new nucleic acid strands that can be several thousand nucleotides in length, provided that the chosen conditions are appropriate.

By using a wash of the modified prima, the procedure can be repeated to make complementary copies of the newly synthesized strands, thus representing identical copies of the matrices initially used. By repeating this process, a reaction cascade is initiated by which the nucleic acid to be detected is amplified. This process can be repeated as many times as desired to obtain the desired detection sensitivity. In cases where the concentration of

-4AT395434B nucleic acid is not extremely low, a duplication is sufficient to make the nucleic acid detectable.

It is also possible to use only a modified primer in the method according to the invention. In this case, however, the duplication will not be as effective as when using at least two

Primers, since no reaction cascade is initiated.

With the method according to the invention, both DNA and RNA can be determined. However, if the nucleic acid to be determined is an RNA, it is more convenient to first prepare a corresponding cDNA copy of the RNA with a reverse transcriptase and then perform the method of the invention.

After the modified primers are incorporated into the copies of the nucleic acids to be determined, the

Reaction mixture with a suitable selective probe molecule, which recognizes the sequence to be determined and their copies, and the hybridization is carried out under conditions that are suitable for the desired hybridization process.

In the hybridization reaction, depending on the choice of the modified primer, either a selective capture probe or a selective detector probe is hybridized with the copies of the nucleic acid to be determined, which is now present in multiple amounts relative to the starting situation.

If the original sample contains the sequence to be detected, the selective probe molecule hybridizes with the newly synthesized copies of the nucleic acid to be determined. A hybrid is formed between the copied molecule in which the modified primer is incorporated and the selective probe molecule. The hybrids formed according to the invention from the hybridization solution in a simple manner with the help of the part of

Separate affinity pair, which is attached either to the capture probe acting as a primer or to the selective capture probe. During fractionation, the hybrids having such a capture moiety will adhere to a solid support with the help of the other portion of the affinity pair. The amount of the selective detector probe or of the primer acting as a detector probe, which adheres to the carrier, can be determined in a manner known per se directly on the carrier material or after the elution in the eluate. The determined amount of

Labeling is a measure of the amount of nucleic acid to be determined.

Prior to fractionation, the solution is optionally diluted to the conditions advantageous for the

Affect affinity pair. The solution is then contacted with the solid support. The support to be used may be, for example, an affinity chromatography column, a filter, a plastic surface or a glass surface. Cheap facilities for carrying out the separation are different

Types of microtiter plates, immersion systems or magnetic particles. However, it is also possible to carry out the separation in test tubes or with granules.

As the carrier material, for example, a natural or synthetic polymer such as cellulose, polyacrylamide, polystyrene, dextran or agarose can be used for the affinity chromatography column. These materials can also be used as suspensions in a test tube. In a preferred embodiment, test tubes are used on the inner surface of which the other part of the affinity pair is bound

The prerequisite for the selected material is that it allows binding of a constituent having affinity for the portion of the affinity pair, is bound to the capture probe primer, or the selective capture probe.

It is not necessary to bind the part or parts of the affinity pair to the primer which acts as a capture probe at the beginning of the polymerization. Also, it is not necessary to use the detectable mark on the as

Capture probe acting primer to bind before the polymerization. They may also be attached or bound after polymerization to the modified primer incorporated into the copies of the nucleic acid to be detected. For example, if the detectable maize is not stable under hybridization conditions, it may also be added after hybridization of the selective capture probe with the copies of the nucleic acid to be detected.

When the detector probes act as modified primers incorporated into the copies of the nucleic acid to be detected, the hybrid can be separated from the reaction mixture by selective capture probes immobilized on solid supports. In such a method, the rate of reaction is limited by the need to hybridize the nucleic acid to be detected and its copies, which incorporate the primers acting as a detector probe, to the selective capture probe immobilized on a solid support.

Therefore, hybridization in solution is preferred in the present invention. However, if the method is performed with an immobilized capture probe, the hybrid formed on the solid support is washed and the amount of label on the support material is measured in a manner known per se.

The examples illustrate the invention.

example 1

Detection of cvtomegalievims DNA with detector probes acting as modified primers

In this example according to the invention, the nucleic acid to be detected is the recombinant plasmid

This contains a 12.3 kb fragment of the cytomegalovirus genome (CMV, AD 169, ATCC VR-538). The two primers used as detector probe (P_a and P_b) (see Fig. 1) have a length of 20 nucleotides and were synthesized in a conventional manner with an automatic DNA synthesizer. They correspond to two regions of CMV-specific insertion, which are 111 nucleotides apart. The two selective capture probes S | and S₂ (See Fig. 1) identify areas in each of the two strands of DNA that lie between the two primers acting as a detector probe. The primers acting as detector probe (P_a and P_b) are with ³²P is marked at its 5 'ends so that its specific activity is 4x10 ^ CPM / pg. The label is in a conventional manner with polynucleotide kinase and gamma³²P-ATP performed; see. Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1982.

The 3 'ends of the capture probes are provided with biotinylated nucleotides. Bio 11-dUTP (BRL) and a terminal transferase (Promega Biotech.) Are used; see. Riley et al., DNA 5 (4) (1986), p. 333,337. The plasmid to be detected is linearized by cleavage with the restriction enzyme EcoRI. DNAPolymerase, Klenow fragment, was purchased from Boehringer-Mannheim and streptavidin-agarose from BRL

With these reagents the following experiment is carried out:

Four different reactions are performed, the 0, 10⁴, 10 ^ and 10⁸ Contain molecules of the plasmid to be detected. This corresponds to 0.2 x 10²θ, 2 x 10 '^⁸ or 2 x 10 -6 moles. Further components of the total reaction volume of 50 μϊ are 2 pmol of the two primers, 0.5 mM of each of the four deoxynucleoside triphosphates (ie dATP, dCTP, dGTP and dTTP), 10 mM Tris-HCl (pH 7.5), 10mM MgCl2, 50mM NaCl and 10mM dithiothreitol. The reaction mixture is heated to 100 ° C. for 2 minutes and then incubated for 5 minutes at 37 ° C. Subsequently, 1 μϊ (1 unit) of DNA polymerase is added. The mixture is incubated for another 10 minutes at 37 ° C. The boil and the subsequent investment of the primer acting as a detector probe and the incubation with the DNA polymerase at 37 ° C is a DNA synthesis cycle. In this experiment, the reaction cycle is either performed only once or repeated 5 or 15 times. After the last reaction cycle, the sample is again heated to 100 ° C. Then 100 pmoles of the selective capture probe and 0.9M NaCl, 20 mM EDTA, 20 mM sodium phosphate ( pH 7.5) and 0.1% Nalriumdodecylsulfat added. The total volume is 100 μϊ. The concentrations given are the final concentrations. The mixture is then incubated for 1 hour at 50 ° C. After this hybridization reaction, 200 μl of a 25% suspension of streptavidin-agarose in 1M NaCl, 20 mM sodium phosphate (pH 7.5) and 1 mM EDTA are added. It is incubated for 15 minutes at 37 ° C in a rotary mixer, so that the biotinylated molecules can bind to the streptavidin-agarose. The agaiosis is spun down briefly and the supernatant is degraded. The agarose is then washed once with buffered IM NaCl solution and twice with a solution containing 150 mM NaCl, 15 mM sodium citrate and 0.2% sodium dodecyl sulphate (pH 8) at 37 ° C. The radioactivity bound to the agarose by the hybrids formed is then determined with a counting device. The isolation and washing process of the DNA hybrids containing the biotinylated markings is carried out as described in GB-A 2 169 403.

The results of the experiment are summarized in Table I. It can be seen from Table I that in a single DNA synthesis cycle, only sufficient radioactivity is incorporated for detection when high starting concentrations of the nucleic acid to be detected are present. However, if 15 DNA synthesis cycles are performed, even very small amounts of this nucleic acid to be detected can be detected. With a large amount of the nucleic acid to be detected and DNA synthesis cycles, the reaction is limited by the amount of primer acting as a detector probe.

Table I

<td>Amount of the pointing nucleic acid to be detected (mol)</td><td colspan="3">³²p.Activity in the collected hybrids ^a) (CPM over background) ®)</td>

<td></td><td> 1</td><td> 5</td><td>15 number of DNA synthesis cycles</td>

<td> 0</td><td> 0</td><td> 0</td><td> 0</td>

<td>2x10-2 °</td><td>ND</td><td>ND</td><td> 650</td>

<td>2xl0-¹⁸</td><td>ND</td><td> 300</td><td> 11000</td>

<td>2xl0⁴⁶</td><td> 700</td><td> 13000</td><td> 36000</td>

-6AT395434B ^a) Average of two measurements

b) ND: not detectable (less than twice as much radioactivity as the mean background activity)

Example 2

Determination of Cvtomegalovirus DNA with Capture Probes as Modified Primers

In this example according to the invention, the capture probes are used as primers Except for the deviations mentioned below, the same reagents as in Example 1 are used. The primers acting as a capture probe (P_a and Pj,) (see Fig. 1) are not denoted by ^²Instead, their 5 'ends are provided with a biotin residue. This chemical modification is carried out in a manner known per se, cf. Chollet and Kawashima, Nucleic Acids Research, 13 (1985), pp. 1529-1541. The two selective probe molecules (Si and S₂) (see Figure 1) are labeled at their 5 'ends in this experiment. They are used as detector probes. Their specific activities are 2 x 10 ^ and 2.5 x 10 ^ cpm / pg, respectively.

The reaction mixtures have the composition described in Example 1. However, the biotinylated primers acting as capture probes are added in 10 times the amount, ie 20 pM per reaction. As described in Example 1, 1.5 or 15 DNA synthesis cycles are performed. The samples are then heated to 100 ° C and 0.5 pM of each of the ^²P-labeled probes (Sj and S₂) are added. The hybridization is carried out under the conditions described in Example 1.

The hybrids are then collected on streptavidin-agarose, washed, and the ^²P activity is determined as described in Example 1. The results are summarized in Table II.

tables

<td>Amount of nucleic acid to be detected (mol)</td><td colspan="2">32p. Activity in the collected hybrids ^a) (CPM over background)¹*)</td>

<td></td><td> 1</td><td>5 15 number of DNA synthesis cycles</td>

<td> 0</td><td> 0</td><td> 0 0</td>

<td>2xl0-²⁰</td><td>ND</td><td>ND 800</td>

<td>2xl0¹⁸</td><td>ND</td><td> 400 13000</td>

<td>2xl0⁴⁶</td><td> 300</td><td> 11000 54000</td>

^a) Average of two measurements ^b) ND: not detectable (see example 1)

Example 3

Detection of cvtomegalovirus DNA from clinical samples with capture probes as modified primers

This example demonstrates that the method of the present invention is suitable for clinical specimen examination. This is accomplished by the detection of CMV from the urine of a child known to be suffering from cytomegalovirus infection. The urine of a healthy child is used as a control. From two samples each containing 10 ml of urine, the total DNA is isolated according to the instructions of Virtanen et al., J. Clin. Microbiol.20 (6) (1984), pp. 1083-1088. The in 20 μ1 H₂Dissolved DNAs are used as the nucleic acid to be detected in reactions carried out according to Example 2. After 10 DNA synthesis cycles, the sample is spiked with the labeled, selective probe molecule. Hybridization is then performed and the hybrids are collected. The DNA from the patient's urine shows markedly increased radioactivity in the hybrids, while that of the healthy child shows only background radioactivity. The actual cpm values ​​are 2300 or 240th

Example 4

Detection of SFV (Semliki Forcst viral RNA with capture probes as modified primers

Example 4 shows that the method according to the invention is also suitable for the detection of RNA. When

Model system is used SFV (Semliki forest virus) RNA

The reagents used are two 5'-biotinylated primers which act as capture probes (see Fig. 2), which are like

-7AT395434B prepared in Example 2, a single 5 '- ^²P-labeled, selective detector probe prepared as described in Example 1, reverse transcriptase (Promega Biotech) and DNA polymerase, Klenow fragment (Boehringer Mannheim).

The first step in the detection of SFV RNA is the synthesis of a cDNA copy. In 20 μM of the reaction mixture, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 10 mM MgClj, 10 mM dithiothreitol, 0.5 mM of each of the four deoxynucleoside triphosphates, 0.5 pg t-RNA, 10 pg SFV RNA, 10 pM primer as a capture probe (P_a) and 100 units of reverse transcriptase. This reaction mixture is incubated at 37 ° C for 15 minutes. The mixture is then heated at 100 ° C for 5 minutes and cooled at ambient temperature. The reaction mixture is then washed with 50 μM of a solution containing 10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 10 mM MgCl 2,

10 mM dithiothreitol, 10 pM of the capture probe primer (P_b) and with 0.5 mM of each of the four

Deoxynucleoside triphosphates added The temperature is raised to 37 ° C. After 5 minutes, 1 unit of DNAPolymerase is added. It is incubated for 10 minutes and the reaction mixture is heated to 100 ° C for 5 minutes. The reaction mixture is then cooled to 37 ° C. In total, 5 DNA synthesis cycles are carried out. After a final denaturation step, 0.1 pmol (1.2 x 10 ^ cpm) of the selective detector probe is added to 80 μl of lMNaCl, 50 mM EDTA, 50 mM sodium phosphate (pH 7.5) and 0.1% sodium dodecyl sulfate. The solution is incubated for 2 hours at 55 ° C the hybrids are collected and washed as described in Example 1.

As a negative control for the reactions, an identical sample is treated in the same way, but without the addition of the reverse transcriptase. The sample in which a cDNA20 copy was made from the reverse transcriptase RNA gives a ^²P-activity of 420 cpm in the captured hybrids, while the negative control gives only 50 cpm

Example 5

Comparison of different methods for the detection of amplified DNA

In this example, three methods for detecting amplified DNA are compared. It will be the

Reagents and the amplification method of Examples 1 and 2 used However, as selective capture or detector probes, M13 clones are used which recognize about 100 nucleotides between the primers.

The M13 clones are usually obtained by subcloning a restriction fragment of the recombinant plasmid pBR322 / CMVHindIIIL in the phage vector M13mpl0in. Dieal selective capture probes to be used with M1 3 clones are modified with biotin using Photoprobe® biotin (Vector Laboratories). The M13 clones to be used as selective detector probes are labeled with²Labeled P-dCTP using DNA polymerase I (Klenow fragment) and primer extension such that the specific activity is 2 x 10 «cpm / pg; see. Hu and Messing, Gene 17 (1982), pp. 271-277.

Now 10 duplication cycles of 3 xl (r molecules (0.5 x 10 *¹^ Mol) of the linearized plasmid pBR322 / CMV HindlH L. For proof according to the method 1 and 2 in each case 2 pmol of the 32p. labeled, acting as a detector probe primer (P_a and P_bThe amplification method is carried out according to Example 1. For the detection according to Method 3, 25 pmol of the biotinylated primer acting as a capture probe is used in the amplification process. The reaction is carried out as described in Example 2.

Results of the detection method 1:

Use of a selective capture probe for collecting the hybrids formed in solution with the copies of the nucleic acid to be detected.

In this detection method, biotinylated selective Ml 3 capture probes are used to collect the ver45 diverse DNA fragments. After the final amplification cycle, the sample mixture is heated to 100 ° C with 2x10 ^ molecules of the biotinylated selective initial probes and NaCl (0.6 M). , EDTA (5 mmol) sodium phosphate (20 mmol) and SDS (0.1%). The final concentrations are given in a final volume of 100 μl. The reaction mixture is incubated for 2 hours at 65 ° C. The hybrids formed are separated on streptavidin-agarose as described in Example 1, but additionally washed twice each for 1 minute at 50 ° C with 15 mM NaCl, 1.5 mM sodium citrate and 0.2% SDS. The radioactivity of the separated hybrids is measured.

Detection method 2:

Use of selective capture probes immobilized prior to hybridization with the copies of the nucleic acid-immobilized probes to be detected.

In this method, immobilized, selective M13 probe molecules are used to capture the amplified DNA. After the last reprocessing cycle, the samples are heated to 100 ° C

-8AT395434B with NaCl (0.6 M), sodium citrate (60 mM), Ficoll® (0.02%), polyvinylpyrrolidone (0.02%), bovine serum albumin (0.02% and denatured herring sperm DNA (0/2 mg As indicated, the final concentrations are in a final volume of 100 μϊ Then each sample is spiked with a nitrocellulose filter disk containing 5χ 10 * Moleküle molecules of each of the selective probe molecules according to the method described in U.S. Patent 4,486,539 immobilized methods are described. The mixture is incubated with filters at 65 ° C for 2 or 18 hours. After hybridization, filters are washed twice each at 50 ° C for 20 minutes with 15 mM NaCl, 1.5 mM sodium citrate, and 0/2% SDS, and the radioactivity bound to the filters is measured.

Detection method 3:

Use of a selective detector probe for detection.

With this method, ^²P-labeled, selective M13 detector probes used to detect the amplified DNA. The hybrids are separated using primers which are biotinylated at their 5 'ends in accordance with Example 2, acting as starting probes. The amplified samples are heated to 100 ° C and with 2x10® molecules (2 χ 10⁵ cpm) of each of the ^²P-labeled, selective probe molecules as in detection method 1 with salts. The hybridization and the separation of the hybrids formed are carried out according to detection method 1.

The results obtained in the three detection methods are summarized in Table ΙΠ and compared.

Methods 1 and 3 have the advantage of a higher rate of hybridization in solution compared to filter hybridization in Method 2. The highest ³²P activity is obtained in Method 3 because the selective detector probe has many ³²Contains P atoms per molecule.

Table III

<td>method</td><td>primer</td><td>selective M13 probe molecule</td><td>hybridization Time Hours.</td><td>32p activity in the hybrid molecules (cpm)^a)</td>

<td> 1</td><td>³²P-labeled primer as a detector probe</td><td>biotinylated capture probe</td><td> 2</td><td> 870</td>

<td> 2</td><td>ditto.</td><td>immobilized capture probe</td><td> 2 18</td><td> 43 920</td>

<td> 3</td><td>biotinylated Primer as a capture probe</td><td>³²P-labeled detector probe</td><td> 2</td><td> 7800</td>

^a) Mean of three measurements (cpm over background)

Example 6

Reproduction of cvtomegalovirus DNA with biotin-labeled primers acting as a detector probe and indirect determination with styptavidin-horseradish peroxidase

In this example, amplification of the CMV plasmid (pBR322 / CMV HindIII L) with the detector probe biotinylated primers (P_a and P | j) according to Example 2. The M13 clones described in Example 5 are modified with sulfone groups and used as selective capture probes. The hybrids formed are separated in the wells of microtiter plates coated with disulfone-modified DNA-labeled antibodies. The detection of the hybrids formed is finally carried out with a streptavidin-horseradish peroxidase conjugate which detects the biotin residues of the primer molecules.

The M13 clones are modified by a sulfonation reaction. The reagents and procedures recommended by Orgenics Ltd (Yavne, Israel) are used. Polystyrene microtiter plates (Nunc, Denmark) are coated with 10 μg / ml IgG in 10 mM sodium carbonate buffer (pH 9.6) overnight at 4 ° C.

-9AT395434B

The IgG was obtained by purifying a monoclonal antibody against sulfone-modified DNA (Orgenics Ltd.).

Reaction mixtures contain 3 × 10⁵ Molecules of the linearized plasmid pBR322 / CMV HindIII L or controls without plasmid and 25 pmoles of each of the biotinylated primers (P_a and P ^) subjected to duplication cycles under the conditions described in Example 1. The samples are heated to 100 ° C and then each with 2 x 10 'molecules of each sulfonated, selective capture probes together with the reagents specified in Example 5 for the method I added

The mixture is incubated for 2 hours at 65 ° C, diluted with 100 μΐ 02% Tween 20 and transferred to the coated microtiter plates The hybrids calf 2 hours at 37 ° C to the wells of the microtiter plate bound. The reaction mixture is discarded and the wells are washed 3 times each with 0.1% Tween 20 in 0.15 molar sodium chloride, 20 mM sodium phosphate (pH 7.5) (PBS). Add 200 μM of a streptavidin-horseradish peroxidase conjugate (Amersham, UK) diluted 1: 2000 in a 1% bovine albumin albumin solution with 0.1% Tween 20 in PBS. Microtiter plate wells are incubated at 37 ° C for 45 minutes this way is washed four times. Then 200 μΐ of a substrate solution consisting of 0.46 mg / ml o-phenylenediamine, 0.01% H₂O2 in 0.1 M sodium acetate buffer (pH 5.0) was added. After 15 minutes at 22 °, the reaction is stopped by addition of 50 μM 2N H 2 SO 4 and the absorbance of the colored reaction product at 492 nm is measured with a photospectrometer. These separation and detection methods have already been described by Syvänen et al in Nucleic Acids Res. 14 (1986), pp. 5037-5048. The results of the experiment are summarized in Table IV 3 x 10 $ molecules (0.5 x 10⁴⁸ Mol) of the plasmid to be detected are clearly detectable after 10 duplication cycles.

Table IV

<td>Amount of nucleic acid to be detected (mol)</td><td>Absorption at 492 nm ^a)</td>

<td>0.5x10⁴⁸</td><td> 0,348</td>

<td> 0</td><td> 0,120</td>

^a) Average of three measurements.