RU2332669C2 - Method of human genotype definition on basis of polymorphism in interleukin 1-alpha gene position (c/t) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: claimed method belongs to medicine, biology and biotechnology fields and concerns definition of human genotype on the basis of polymorphism in interleukin-1-alpha gene in position 889(C/T). The method involves use of allele-specific primers with polymerase chain reaction directly in the course of reaction with the help of fluorescent marked samples, the reaction mix including primers for a site with desirable replacement, different for each allele, and primer with sample common to each couple of alleles.

EFFECT: streamlining of method.

2 cl, 1 dwg

Description

The proposed method relates to the field of medicine, biology and biotechnology and can be used to determine the human genotype by polymorphism in the interleukin gene 1-α position 889 (C / T), referring the test sample to heterozygous or homozygous according to one of the allelic variants.

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 kits use one pair of primers for each allele and two samples with different fluorescent labels, depending on the genotype of the allele, the burning of different labels is recorded. ("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 technical result of the invention is to increase the availability of such studies, since the method can be implemented on standard known equipment. It also provides the possibility of using only one labeled sample, which reduces research costs and leads to a decrease in their cost.

This result is achieved by the fact that in the method for determining the human genotype based on the use of allele-specific primers with the registration of PCR results directly during the reaction using fluorescently-labeled samples, the human genotype is determined by polymorphism in the interleukin gene 1-α position 889 (C / T), while the reaction mixture includes primers for the site with the desired replacement, different for each allele, and common for two alleles, a primer and a sample of the following form:

Primer (C allele): 5'-CGC GTA ATA GTA ACC AGG CAA CAC-3 '

Primer (T allele): 5'-CGC GTA ATA GTA ACC AGG CAA CAT-3 '

General primer (C, T alleles): 5'-GAT ATG CCC AAG GTG TGT CTT CTG-3 '

Total sample (C, T alleles): (BHQ1) -5'-TTA CCT CCC GGG GC (FdT) TGC ACA CAC C-3 '- (P),

where BHQ1 is a dark fluorescence quencher attached to the 5'-terminal nucleotide.

FdT - FAM fluorescent dye 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 (composition of the 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.

The drawing shows an example of graphs of DNA accumulation obtained using a detecting amplifier for allele-specific reactions. 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 (the curves are shown on a linear scale). Graph B is the result of a study of a homozygous sample (the curves are shown on a logarithmic scale). Graph B is the result of a study of a heterozygous sample (the curves are depicted on a linear scale). Graph D is the result of studying a heterozygous sample (the curves are shown on a logarithmic scale).

PCR is carried out in separate tubes for each variant of the allele. In order to distinguish between two alleles, use a primer on the site with the desired replacement, which is different for each allele, and a common primer for the two alleles.

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

A DNA sample is added to this reaction mixture and real-time PCR is carried out, the signal is recorded using detecting amplifiers.

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

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

II. The difference between Ct (ΔCt) for two alleles is determined. 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 from 0 to 2 cycles. Such a sample should be interpreted as heterozygous for the studied polymorphism;

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

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 samples.

- 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.

For research, the use of human blood is recommended, from which DNA is isolated using a set of reagents for DNA extraction, including:

- ficoll-urographin (a reagent for the isolation of lymphocytes);

- lysing solution (cell lysis reagent);

- precipitation reagent (nucleic acid precipitation reagent);

- washing solution No. 1 (reagent for removing extraneous

impurities from the nucleic acid preparation and inactivation of RNases);

- Wash solution No. 2 (reagent to remove impurities from the nucleic acid preparation);

- a buffer for dissolution (a reagent for dissolving nucleic acids, free of RNases).

Claims (2)

1. The method of determining the human genotype by polymorphism in the interleukin gene 1-α position 889 (C / T), based on the use of allele-specific primers with the registration of PCR results directly during the reaction using fluorescently-labeled samples, characterized in that the genotype human is determined by polymorphism in the gene, while the reaction mixture includes primers for the site with the desired replacement, different for each allele, and common for the two alleles, a primer and a sample of the following type: Primer (allele C): 5'-CGCGTAATAGTAACCAGGCAACAC-3 ' Primer (T allele): 5'-CGCGTAATAGTAACCAGGCAACAT-3 ' Total primer (alleles C, T): 5'-GATATGCCCAAGGTGTGTCTTCTG-3 ' Total sample (C, T alleles): (BHQ1) -5'-TTACCTCCCGGGGGC (FdT) TGCACACACC-3 '- (P), where BHQ1 is a dark fluorescence quencher attached to the 5'-terminal nucleotide, FdT is a PAM fluorescent dye attached to nucleotide T and after the end of the amplification program the average value of the threshold cycle (Ct) for PCR duplicates is determined, then the difference between Ct (ΔCt) for two alleles is determined, and if ΔCt is between kinetic curves describing two reactions is in the range from 0 to 2 cycles, then such a sample should be interpreted as heterozygous for the studied polymorphism, and if ΔCt between the kinetic curves describing the two reactions is in the range from 4 cycles and above, then such a sample with eduet interpreted as a homozygous genotype in this case corresponds to the allele a smaller Ct. 2. The method for determining the human genotype according to claim 1, characterized in that the following standard components are included in the reaction mixture: a mixture of four types of deoxyribonucleotide triphosphates; reaction buffer (composition of the 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.

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