RU2445368C2 - METHOD FOR HUMAN GENOTYPE DETERMINATION BY 5382 GENE BRCA1 (5382insC) POSITION POLYMORPHISM - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention may be used for human genotype determination by 5382 gene BRCA1 (5382insC) position polymorphism. The method consists in using allele-specific primers with real-time PCR values recording with using fluorescence marked probes. A reaction mixture contains the primers different for each allele, the primer and the probe common for two alleles, and also the common fluorescent probe.

EFFECT: invention provides cutting expenses for genetic researches and making them more available due to the use of standard equipment and only one marked probe.

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Description

1. Field of technology.

The proposed method relates to the field of medicine, biology and biotechnology, and can be used to determine the human genotype by polymorphism at position 5382 of the BRCA1 gene (5382insC) with assignment of the test sample to heterozygous or homozygous according to one of the allelic variants.

Breast cancer (BC) is the most common cancer in women: it affects at least one in ten women in developed countries and takes a leading position in mortality due to imperfections in early diagnosis. Individual risk of breast cancer is often associated with burdened heredity, i.e. oncological mutations associated with the risk of breast cancer. Speaking about the identification of persons at risk for the development of breast cancer, it is necessary to take into account the marker genes identified and characterized by now, mutations in which lead to the development of breast cancer and ovarian cancer. One of these genes is the BRCA1 gene.

2. The prior art.

From the literature data it is known that in the residents of Russia approximately 20% of breast cancer that occurred at a young age (up to 40-50 years) due to unfavorable heredity are explained by mutations in the BRCA1 gene. For a number of mutations in the BRCA1 gene, a stable association with an increase in the risk of developing breast cancer over the course of life by 50-80% is shown. One of these mutations is polymorphism at position 5382 of the BRCA1 gene (5382insC).

A widespread method for determining the type of nucleotide located at a specific DNA site is based on the use of allele-specific primers with registration of PCR results at the end of the reaction by electrophoresis.

Various methods are known for determining the type of nucleotide located at a specific DNA site, based on the use of allele-specific primers with the registration of PCR results directly during the reaction using fluorescently-labeled samples (oligonucleotides) (Andreas R. Tobler at all, "THE SNPlex Genotiping System: A Flexible and Scalable Platform for SNP Genotyping ", Journal of Biomolecular Techniques, V.16, issue 4, Desember 2005).

When using PCR with recording the results during the reaction, various methods are known for determining the genotype of the test sample.

For example, Applied Biosystems production kits use one pair of primers for each allele and two probes with different fluorescence labels; depending on the allele genotype, different labels burn up ("TaqMan SNP Genotyping Assays", Applied Biosistems, Prodakt Bulletin, USA, 06/2006 ) The disadvantage of the method used in these sets is its high cost.

The present invention makes more accessible and cheaper such studies, thanks to the use of standard equipment and the use of only one labeled probe.

3. Disclosure of the invention.

The technical result, the achievement of which the invention is directed, is to increase the availability of such studies, since the method can be implemented on standard known equipment. It also provides the ability to use only one labeled probe, which reduces research costs.

The specified result is achieved by using allele-specific primers in the PCR formulation for the site with the desired substitution, which are different for each allele, and the primer and probe of the following nucleotide composition common to two alleles:

Primer (lack of insertion C)

BRCA1-53821 5'-AAg CgA gCA AgA gAA TTC CAg-3 '

Primer (insertion C)

BRCA1-53822 5'-AgC gAg CAA gAg AAT TCC CA-3 '

Common primer

BRCA1-53823 5'-AgA ACC TgT gTg AAA gTA TCT AgC ACT g-3 '

Common probe

BRCA1-5382t (BHQ1) -5'-TTC TCC CCT T (FdT) g CAg AgA Tgg gCC gC-3 '- (P),

where BHQ1 is a dark fluorescence quencher attached to the 5'-terminal nucleotide, and FdT is a fluorescent dye FAM attached to nucleotide T.

Additionally, the following standard components are included in the reaction mixture:

- a mixture of four types of deoxyribonucleotide triphosphates;

- reaction buffer (100 mm Tris-HCl (pH 8.8 at 25 ° C), 500 mm KCl, 0.8% P40, 20 mm MgCl ₂ );

- bidistilled water;

- Taq polymerase;

- mineral oil;

- paraffin.

Figure 1 shows an example of graphs of DNA accumulation obtained for allele-specific reactions using a detecting amplifier. The axis of coordinates “x” is the fluorescence level, “y” is the cycle number. Graph A is the result of a study of a homozygous sample (curves are shown on a linear scale). Graph B is the result of a study of a heterozygous sample (the curves are depicted on a linear scale).

NDP is carried out in separate tubes for each variant of the allele. In order to distinguish between two alleles, a primer is used for the site with the initial substitution, which is different for each allele, and a common primer for the two alleles.

As a result of this choice of primers and probe, one can judge the genotype of the test sample by the shape and location of the DNA accumulation curves.

4. The implementation of the invention.

It is recommended to use blood, scrapings of buccal epithelium, and biopsy specimens of human soft tissues as a material for research. Isolation of DNA from biomaterial is performed using a set of reagents for DNA isolation (not the subject of this patent).

A DNA sample is added to this reaction mixture and real-time PCR is performed, the signal is recorded using a detecting amplifier.

The genotype (allelic composition) of the test sample is determined after the end of the amplification program by the ratio of the threshold cycles for each PCR product (the threshold cycle is the number of the PCR temperature cycle at which the detecting amplifier registered the appearance of a specific signal for this type of reaction product) as follows:

1. Determine the average value of the threshold cycle (Ct) for PCR duplicates.

2. Determine the difference between Ct (ΔCt) for two alleles.

For the test sample, the following options for the relative location of the kinetic curves are possible:

A) ΔCt between the kinetic curves describing the two reactions is in the range of 4 cycles and above. Such a sample should be interpreted as homozygous for the studied polymorphism. The genotype in this case corresponds to an allele with a lower Ct.

B) ΔCt between the kinetic curves describing the two reactions is in the range from 0 to 2 cycles. Such a sample should be interpreted as heterozygous for the studied polymorphism.

To apply this method on an industrial scale, the components of the reaction mixture can be produced in a kit, ready for use:

- tubes with a mixture for amplification, sealed with paraffin, including reaction buffer, deoxyribonucleotide triphosphates, primers, fluorescent probes;

- tubes with a solution of Taq polymerase;

- test tubes with mineral oil.

When using this kit, Taq polymerase solution, mineral oil and a prepared DNA sample are added to the tubes with the mixture for amplification.

Claims (1)

A method for determining the human genotype by polymorphism at position 5382 of the BRCA1 gene (5382insC), based on the use of allele-specific primers with real-time PCR results, characterized in that primers that are different for each allele are used, a common primer for the two alleles and a common fluorescent probe of the following nucleotide composition:
Primer (lack of insertion C)
BRCA1-53821 5'-AAg CgA gCA AgA gAA TTC CAg-3 '
Primer (insertion C)
BRCA1-53822 5'-AgC gAg CAA gAg AAT TCC CA-3 '
Common primer
BRCA1-53823 5'-AgA ACC TgT gTg AAA gTA TCT AgC ACT g-3 '
Common probe
BRCA1-5382t (BHQ1) -5'-TTC TCC CCT T (FdT) g CAg AgA Tgg gCC gC-3 '- (P),
where BHQ1 means attached to the 5'-terminal nucleotide dark quencher of fluorescence, and FdT is a fluorescent dye FAM attached to nucleotide T,
and after PCR using these primers, the average threshold cycle (Ct) value for PCR duplicates is determined and the difference between Ct (ΔCt) for two alleles is fixed, and if ΔCt between the kinetic curves describing the two reactions is in the range from 0 to 2 cycles, such a sample should be interpreted as heterozygous for the studied polymorphism, and if ΔСt between the kinetic curves describing the two reactions is in the range of 4 cycles or higher, then such a sample should be interpreted as homozygous for tivity polymorphism and the genotype in this case corresponds to the allele a smaller Ct.

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