RU2240350C1 - Method for identifying gene mutation and polymorphism - Google Patents

Abstract

FIELD: medicine, medicinal genetics.

SUBSTANCE: invention can be used in molecular diagnosis. The proposed method for identification of gene mutations and polymorphisms is carried out by run of allele-specific polymerase chain reaction in the presence of depositing oligonucleotides that are complementary to polymorphous primers. Invention provides simplifying the record for optimization of allele-specific polymerase chain reaction and to enhance reliability of obtained data.

EFFECT: improved identifying method.

2 dwg, 1 ex

Description

The invention relates to medicine, in particular to molecular diagnostics, molecular medical genetics, molecular oncology.

Allele-specific polymerase chain reaction (PCR) is one of the methods for direct detection of known point mutations and single nucleotide polymorphisms. The method is based on the inability of Taq DNA polymerase to amplify a fragment in the presence of a mismatch between the variable nucleotide on template DNA and the 3'-end of one of the oligoprimers (Newton C.R. et al., 1989; Bottema C.D.K. et al., 1990).

However, under suboptimal PCR conditions, fragment amplification often occurs even in the event of such a mismatch. This circumstance very significantly limits the use of allele-specific PCR (Malcolm E.K. et al., 2000).

The following methods are known to increase the reliability of allele-specific PCR (Sarkar G. et al., 1989; Sommer S.S. et al., 1992):

1) increasing the temperature of the annealing of the primers;

2) reduction in the number of PCR cycles;

3) a decrease in the concentration of key PCR components (magnesium chloride, nucleotides, DNA polymerase, primers, the original DNA matrix);

4) adding to the reaction substances that non-selectively increase the specificity of PCR, the so-called “specificity enhancers”: glycerol, X-100 triton, formamide, dimethyl sulfoxide, etc .;

5) the addition of a specific but not elongable primer containing a dideoxynucleotide (ddNTP) at the 3'-end (Orou A. et al., 1995).

Closest to the proposed is a method based on reducing the concentration of primers in the reaction (Sommer SS, Groszbach AR, Bottema CD: PCR amplification of specific alleles (PASA) is a general method for rapidly detecting known singlebase changes. // Biotechniques. 1992 Jan; 12 (1): 82-7). In the described method in order to achieve the specificity of the reaction, a decrease in the absolute concentration of primers in the reaction to 0.05 μM or less is used.

However, this method is far from being able to achieve strict PCR specificity in all cases. In addition, it, like the other methods listed above, has a significant drawback, because significantly reduces the yield of the PCR product.

Thus, in the process of optimization of allele-specific PCR, it is necessary to empirically find a rather narrow range of conditions when, on the one hand, their rigidity does not allow amplification of a non-specific fragment, and on the other hand, the reaction is not completely suppressed. Practice shows that the implementation of this task is fraught with serious, sometimes insoluble difficulties.

The technical result achieved by the invention is as follows: simplification of the optimization protocol for allele-specific PCR, increasing its reliability. This is achieved through the use of reversible deposition of primers using complementary deposition oligonucleotides.

The invention consists in the following.

In the present invention, depositing oligonucleotide sequences complementary to allele-specific primers are added to each of the PCR reactions. This allows you to reversibly deposit part of the primers in the reaction. In this system, there is competition for an allele-specific primer between the depositing oligonucleotide and the DNA template. In the case of complete complementarity between the allele-specific primer and the matrix, immediately after annealing of the primer, it is elongated, which increases the affinity of the primer to the matrix and is the beginning of the synthesis of the amplified fragment (Fig. 1, upper right fragment). In the case of incomplete complementarity between the allele-specific primer and the matrix, when there is a 3'-terminal unpaired nucleotide and the elongation is significantly difficult, the primer has time to dissociate with the matrix, and the depositing oligonucleotide, by binding it, prevents reassociation with the matrix (Fig. 1, lower right ) Thus, non-specific amplification is prevented (Fig. 1, left column). A significant advantage of this approach is that the deposition of primers is reversible, because the process of annealing-melting of primers and depositing oligonucleotides is repeated with each PCR cycle. Due to this, the addition of depositing oligonucleotides at concentrations comparable to the concentration of primers (the ratio of depositing oligonucleotides to primers can vary in the range 0.5: 1 - 5: 1) does not critically affect the kinetics of the reaction and allows using the usual number of PCR cycles to obtain a sufficient amount of product .

An example embodiment of the invention.

At the first stage, the allele-specific PCR of the TNF-α gene fragment containing the 308G / A polymorphism was optimized. To exclude false results, genotypes were also determined using the PCR-RFLP method (restriction fragment length polymorphism) (figure 2). Allele-specific PCR included 2 allele-specific primers [5 '- CAATAGGTTTTGAGGGGCATGG - 3' (primer G); 5 '- CAATAGGTTTTGAGGGGCATGA - 3' (primer A)] for the determination of a polymorphic nucleotide, as well as a common primer: 5 '- CGATGGAGAAGAAACCGAGA - 3'. The main parameters of PCR were as follows: 50 ng of genomic DNA, 0.5 units. modified (heat-activated) Taq DNA polymerase, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 5% glycerol, 1.0 mM MgCl ₂ , 200 μM dNTP, 0.125 μM of each primer, final reaction volume - 10 μ1, 30 cycles (95 ° -35 ", 60 ° -1 ', 72 ° -50"). Rather stringent conditions were found to be optimal for this reaction: high annealing temperature (t = 60 ° С), low concentration of magnesium chloride (1.0 mM), low concentration of primers (0.125 μM) and a limited number of PCR cycles (n = 30). When relaxing any of these parameters, nonspecific amplification was observed (Fig. 2, left column). On the other hand, more stringent PCR conditions led to a complete absence of the product (data not shown).

Reactions under the same conditions were carried out in the presence of complementary deposition oligonucleotides (5 '- СCATGCCCCTCAAAACCTAT - 3' for primer G, 5 '- ТCATGCCCCTCAAAACCTAT - 3' for primer А). The concentration of depositing oligonucleotides was equal to the concentration of polymorphic primers. In both cases, both with and without depositing oligonucleotides, under severe conditions, only a specific reaction was observed. The intensity of the fragments on the polyacrylamide gel after gel electrophoresis was the same.

Further, the same reactions were carried out in parallel under relaxed conditions [a decreased annealing temperature (t = 55 ° C), the concentration of magnesium chloride was 1.5 mM, the concentration of primers was 0.250 μM, and the greater the number of PCR cycles (n = 35)], and in each experience changed both individual of these parameters, and all at the same time (figure 2, the right column). In the traditional protocol of allele-specific PCR under relaxed conditions, a nonspecific reaction was observed in all cases, while the allele-specific PCR in the presence of depositing oligonucleotides remained strictly specific, without a noticeable decrease in the quantitative yield of the reaction product.

The present invention includes a previously unknown approach to increasing the reliability of allele-specific PCR, which allows to increase the efficiency and expand the capabilities of molecular diagnostic methods based on the detection of known single nucleotide polymorphisms and point mutations. The proposed method allows to expand the range of conditions acceptable for allele-specific PCR.

Claims (1)

A method for identifying gene mutations and polymorphisms by means of an allele-specific polymerase chain reaction, characterized in that the allele-specific PCR is carried out in the presence of depositing oligonucleotides complementary to polymorphic primers in a ratio of 1: 0.5-1: 5.

Images