RU2346053C2 - Method of identifying polymorphism r287q in 8 exon gene cytosolic epoxide hydrolase in human beings - Google Patents

Abstract

FIELD: chemistry, biochemistry.

SUBSTANCE: invention relates to the region of molecular biology and can be used in medicine during diagnostics of predisposition to multi-factorial diseases in man. Method of detecting polymorphism R287Q in 8 exon gene cytosolic epoxide hydrolase (EPHX2) with the method PCR-RLFP using direct primer 5'-CTG-TGG-CTC-TTG-GTT-TGA-TC-3' and reverse primer 5'-TTC-ATG-TCC-ATA-GCT-AGG-ATC-3', at the 3'-end to which is introduced a restriction site for endonuclease BamHI, used later for hydrolysis of PCR-of the product is offered. In this case obtaining during fractionating in the agarose gel two fragments of DNA of the size 143 and 21 p.o are verified as homozygous genotype of the wild type 287RR, three fragments size 164, 143 and 21 p.o - as the heterozygous genotype 287RQ, and one of the fragments size 164 p.o - as homozygous mutant genotype 287QQ. Offered is a method of genotyping and by achieved with minimal material costs in the laboratory, with standard equipment for carrying out polymeric chain reactions.

EFFECT: developing an effective and economic method of detecting polymorphism R287Q in 8 exon gene cytosolic epoxide hydrolase.

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Description

The invention relates to the field of molecular biology and medicine, namely to DNA diagnostics of a predisposition to multifactorial diseases in humans.

The study of the genetic polymorphism of the nitosol epoxide hydrolase gene (soluble epoxide hydrolase, EPHX2} in cardiovascular diseases is the subject of intensive research in modern cardiology (Fornage M., Boerwinkle E., Doris PA, Jacobs D., Liu K., Wong ND Polymorphism of the soluble epoxide hydrolase is associated with coronary artery calcification in African-American subjects // Circulation. - 2004. - Vol.109. P.335-339; Lee CR, North KE, Bray MS, Fornage M., Scubert JM, Newman JW , Hammock BD, Couper DJ, Heiss G., Zeidin DC Genetic variation in soluble epoxide hydrolase (EPHX2) and risk of coronary heart disease: The Atherosclerosis Risk in Communities (ARIC) study // Hum. Mol. Genet. - 2006. - Vol.15. - P. 1640-1649).

To date, the nucleotide sequence of the EPHX2 gene (NCBI GeneID: 2053; OMIM: 132811) has been completely deciphered, its structural organization has been described, and numerous polymorphic regions of the gene have been identified - the so-called single nucleotide polymorphisms (SNPs). However, functionally significant SNPs located in the coding part of the EPHX2 gene (Przybyla-Zawislak BD, Srivastava P.K., Vázquez-Matias J., Mohrenweiser HW, Maxwell JE, Hammock BD, Bradbury JA, Enayetallah A, became the most attractive objects for genetic studies , Zeidin DC, Grant DF Polymorphisms in human soluble epoxide hydrolase // Mol. Pharmacol. - 2003. Vol.64. - P.482-490; Sato K., Emi M., Ezura Y., Fujita Y., Takada D ., Ishigami T., Umemura S., Xin Y., Wu LL, Larrinaga-Shum S., Stephenson SH, Hunt SC, Hopkins PN Soluble epoxide hydrolase variant (Glu287Arg) modifies plasma total cholesterol and triglyceride phenotype in familial hypercholesterolemia: intrafamilial association study in an eight-generation hyperlipidemic kindred // J. Hum. Genet. - 2004. Vol. 49. - P.29-34). One of these functionally important polymorphic sites of cytosolic epoxyhydrolase is the nucleotide substitution of G860A in the exon 8 of the gene (NCI dbSNP rs751141 reference sequence; Ensembl db 35239), accompanied by the amino acid substitution of arginine for glutamine at the 287 position of the polypeptide chain of the enzyme, leading to a decrease in its activity.

An analysis of the literature shows that to date, high-tech and high-performance methods have been developed for identifying R287Q polymorphism of the coding part 8 of the exon of the EPHX2 gene: automatic sequencing (Sandberg M., Hassett C., Adman ET, Meijer J., Omiecinski CJ Identification and functional characterization of human soluble epoxide hydrolase genetic polymorphisms // J. Biol. Chem. - 2000. - Vol.275. - P.28873-28881; Saito S., Iida A., Sekine A., Eguchi C „Miura Y., Nakamura Y. Seventy genetic variations in human microsomal and soluble epoxide hydrolase genes (EPHX1 and EPHX2) in the Japanese population // J. Hum. Genet. - 2001. - Vol. 46. - P.325-329; Przybyla-Zawislak BD , Srivastava PK, Vázqucz-Matias J., Mohrenweiser HW, Maxwell JE, Hammock B. D., Bradbury JA, Enayetallah AE, Zeidin DC, Grant DF Polymorphisms in human soluble epoxide hydrolase // Mol. Pharmacol. - 2003. Vol.64. - P.482-490) and DNA chip method (Fornage M., Lee CR, Doris PA, Bray MS, Heiss G., Zeidin DC, Boerwinkle E. The soluble epoxide hydrolase geneharbors sequence variation associated with susceptibility to and protection from incident ischemic stroke // Hum. Mol. Genet. - 2005.- Vol. 14. - P.2829-2837).

Closest to the claimed method for identifying R287Q EPHX2 gene polymorphism is the allelic discrimination method using 5 'nuclease analysis (TaqMan, Applied Biosystems) (Fornage M., Boerwinkle E., Doris PA, Jacobs D., Liu K., Wong ND Polymorphism of the soluble epoxide hydrolase is associated with coronary artery calcification in African-American subjects // Circulation. - 2004. - Vol.109. - P.335-339), based on the resonant transfer of fluorescence energy (Livak KJ Allelic discrimination using fluorogenic probes and the 5 'nuclease assay // Genet. Anal. 1999. - Vol.14. - P.143-149). The principle of the phenomenon is that if two fluorophores are located in close proximity to each other and the emission spectrum of the first coincides with the absorption spectrum of the second (quencher), then when the first is excited, instead of fluorescence, energy will be transferred to the quencher. With increasing distance between the fluorophore and the quencher, fluorescence is restored. Resonance quenching of fluorescence in a TaqMan system allows controlling the kinetics of PCR amplification directly during the reaction. For detection, a probe incapable of lengthening, carrying a fluorophore, and a quencher complementary to the middle part of the amplified fragment are used. When Taq polymerase destroys it during the PCR reaction due to exonuclease activity coupled with DNA synthesis, the fluorescent group goes into solution and moves away from the quencher. The use of two allele-specific probes with different fluorescent labels (in particular, FAM-TAGGACCcGGTAACC and VIC-CTAGGACCtGGTAACC) allows us to differentiate homo (287RR and 287QQ) and heterozygotes (287RQ) using a special ABI7900 instrument (Applied Biosystems for Detection) Sequence Detection System Software).

This method has a fairly high sensitivity and specificity, but requires special expensive equipment and specific reagents, which makes its use virtually impossible in standard PCR laboratories in Russia.

Methods for identifying SNP R287Q of the EPHX2 gene using the polymerase chain reaction and analysis of restriction fragment length polymorphism (PCR-RFLP) are not described in the literature. Thus, there is a need to create a quick, simple, inexpensive, sensitive and specific method that could be introduced into a standard clinical laboratory as a routine method for genotinizing the R287Q polymorphism of the EPHX2 gene. Such a method is provided by the present invention.

The objective of the invention is to reduce the material costs for genotyping a single nucleotide polymorphism R287Q of the EPHX2 gene by developing a method for its identification using PCR-RFLP.

The invention is illustrated by two figures:

Pa figure 1 shows a fragment of genomic DNA (plot 7 of the intron and 8 exon) of the EPHX2 gene (orientation 5 '→ 3'). The gray areas of the selected primers are highlighted. The ↑ symbol indicates the site of DNA cleavage with the endonuclease BamHI (recognition site G ↑ GATCC). The unpaired nucleotide at the 3'-end of the antisense primer is underlined (nucleotide G is replaced by A).

Figure 2 shows the electrophoretic separation of PCR restriction products of the EPHX2 gene. On the left are the sizes (bp) of fractionated DNA fragments. Above are the serial numbers of the analyzed samples of 24 people. Sample 1 is a marker (an unrestricted amplicon measuring 164 bp), samples 2, 3, 4, 5, 6, 9, 10, 11, 12, 13, 14, 17, 18, 19, 21, 22, and 24 ( fragment 143 bp, fragment 21 bp not visualized) - homozygous genotypes for the wild-type 287RR allele; sample 16 (one fragment 164 bp in size) - homozygous genotype for the mutant allele 287QQ; samples 7, 8, 15, 20, and 23 (fragments 164 and 143 bp, fragment 21 bp are not visualized) - heterozygous genotypes 287RQ.

The method of genotyping is based on the methodology of PCR-mediated site-directed mutagenesis proposed by Eiken H.G. et al. (Eiken H.G., Odland E., Ramos C. et al. Application of natural and amplification created restriction sites for the diagnosis of PKU mutations // Nucleic Acids Res. - 1991. - Vol.19. - P.1427-1430).

The proposed method is as follows:

First, based on the nucleotide sequence of the cytosolic epoxide hydrolase gene, using the GeneFisher 1.3 program (http://bibiserv.techfak.uni-bielefeld.de), a pair of primers was selected that flanked the region of interest 8 of the EPHX2 exon, having the following structure:

F: 5'-CTGTGGCTCTTGGTTTGATC-3 '(SEQ ID NO 1)

R: 5'-TTCATGTCCATAGCTAGGATC-3 '(SEQ ID NO 2) The amplified region of DNA was chosen so that the 3'-end of the antisense primer was directly adjacent to the mutant site (Fig. 1). In the antisense primer, we changed the penultimate nucleotide from C to T (in figure 1, in the antisense chain G to A, respectively, the nucleotide complementarity) so that, in combination with the nucleotide of the mutant site, a restriction site for BamHI endonuclease (recognition site G ↑ GATCC) is formed at this place . Thus, PCR products of the normal and mutant alleles differ in the presence of this induced restriction site.

When developing a method for genotyping R287Q polymorphism of the EPHX2 gene, genomic DNA samples isolated from frozen venous blood using the standard two-stage phenol-chloroform extraction method were used (Maniatis T. et al., 1984). The primers were synthesized in the NGO Litekh (Moscow). Amplification was performed on a multichannel thermocycler "Tertsik" (ZAO NPF "DNA-Technology", Moscow). PCR parameters were as follows: "hot start" for 4 min at 95 ° C, then 36 cycles of amplification in 95 ° C mode - 30 sec; 55 ° C - 40 sec; 72 ° C for 30 seconds, the final cycle of DNA elongation for 3 minutes at 72 ° C. The size of the EPHX2 gene fragment amplified during PCR was 164 nucleotide pairs (bp). The nucleotide sequence of the EPHX2 gene amplification product was determined using an ABI PRISM 310 Genetic Analyzer using the BigDye Termination Kit 1.1 (Applied Biosystems, United States).

In the preparation of solutions for PCR, imported highly purified reagents (Ultra pure and Biotechnology Grade) were used. PCR was performed in 12 μl of the reaction mixture containing 0.5 μl of a sample of genomic DNA. The amplification mixture included: 67 mM Tris-HCl pH = 8.8, 16.6 mM ammonium sulfate, 6.7 μM EDTA, 1.6 mM MgCl ₂ , 1 mm β-mercaptoethanol, 1 mm each dNTPs (dATP, dCTP , dTTP and dGTP), 15 nM of each of the primers and 1U (1 unit of activity) Taq polymerase. To prevent evaporation of the amplification mixture and the formation of condensate, 30 μl of mineral oil was layered onto the reaction mixture.

Successful PCR was monitored by electrophoresis of amplification products on a 2% agarose gel. Detection of R287Q EPHX2 gene polymorphism was carried out by treating the BamHI endonuclease 6U amplicon (Sibenzim LLC, Novosibirsk) according to the protocol described by the enzyme manufacturer. The restriction mixture was thermostated overnight at 37 ° C. After incubation, DNA fragments were fractionated by electrophoresis in 2.5% agarose gel prepared on the basis of TBE buffer (0.089 M Tris-HCl, 2 mm EDTA, 0.089 M boric acid) s 0.01% ethidium bromide. Fractionation of DNA fragments was carried out in a SE-2 horizontal electrophoresis chamber (Helikon LLC, Moscow) for 80 min at a voltage of 200 V. Phage λ DNA hydrolyzed with PstI endonuclease was used as a molecular weight marker. The separated DNA fragments were visualized on a transilluminator using a GDS-8000 computer video recorder (UVP, USA). Documentation and image processing of electrophoretic gels was carried out using the Labworks analytical package.

On the day of demonstrating the effectiveness of the developed method for identifying R287Q polymorphism of the EPHX2 gene, 23 volunteers were genotyped. The results of electrophoretic separation of PCR restriction products of the R287Q polymorphism of the EPHX2 gene are presented in FIG. 2. In the presence of the wild-type 287R allele, there is a recognition site for the BamHI endonuclease, which cleaves a 164 bp amplicon. into two fragments 143 and 21 bp (21 bp fragment is not visualized). In the presence of a mutation (287Q EPHX2 allele), the recognition site for the BamHI endonuclease is lost: G ↑ GATCC (287R) (AGATCC (287Q), as a result of which the amplicon remains unsplit by this restriction enzyme and is visualized as 1 fragment of 164 bp in heterozygous Owing to the presence of both allelic variants of the gene, 287RQ EPHX2 carriers contain all three DNA fragments: 164, 143, and 21 bp (Fig. 2). As can be seen from Fig. 2, a sufficiently high quality of visualization of the DNA fragments made it possible to effectively detect three variants of polymorphism genotypes Arg287Gln in ge e ERNH2 all 23 samples analyzed.

Thus, the presented results show that the proposed method can effectively identify the R287Q polymorphism in the cytosolic epoxide hydrolase gene using PCR-mediated site-directed mutagenesis and subsequent restriction analysis of amplification products. Due to the low cost of the analysis, due to the low price of BamHI endonuclease Sibenzym LLC, the proposed method of genotyping can be applied in any PCR laboratory with minimal material costs.

Claims (1)

A method for identifying R287Q polymorphism in exon 8 of a human cytosolic epoxide hydrolase gene, including preparing a DNA sample, amplifying a polymorphic site of a gene region using PCR with specially selected primers, analyzing the PCR product and drawing conclusions based on its results, characterized in that primers with SEQ are used ID NO: 1 (forward primer) and SEQ ID NO: 2 (reverse primer), where a restriction site for BamHI endonuclease was introduced at the 3'-end of the reverse primer by replacing one nucleotide, the PCR product was subjected to hyd olysis with BamHI endonuclease and fractionated on an agarose gel, wherein two fragments of 143 and 21 bp were obtained. verified as the homozygous genotype of wild-type 287RR, three fragments of 164, 143 and 21 bp in size. - as a heterozygous genotype 287RQ, and one fragment of 164 bp in size - as a homozygous mutant genotype 287QQ.

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