RU2807808C1 - Set of primers and probes for genotyping of rs12566098 (cg) polymorphic locus of serbp1 gene in human using real-time pcr - Google Patents

Abstract

FIELD: biotechnology; molecular genetic diagnostics; medicine.

SUBSTANCE: invention can be used in determining the hereditary predisposition to the development of diseases associated with carriage of rs12566098 polymorphic variant (C>G) of SERPINE1 gene of mRNA-binding protein 1. The following is proposed: a set of primers for genotyping of rs12566098 polymorphic locus (C>G) of SERBP1 gene in a human using real-time PCR.

EFFECT: invention makes it possible to expand the arsenal of tools for genotyping of polymorphic variants of SERBP1 gene and increase the accuracy of genotyping.

1 cl, 1 dwg

Description

The invention relates to the field of biotechnology, molecular genetic diagnostics, in particular to the assessment of single nucleotide polymorphism rs12566098 (C>G) SERPINE1 gene mRNA-binding protein 1 molecular genetic research method.

GeneSERBP1(Gene ID: 26135) is located on chromosome 1p31.3. According to SNPinfo Web Server/LD TAG SNP Selection [https://snpinfo.niehs.nih.gov/snpinfo/snptag.html] polymorphic variant rs12566098 geneSERBP1 is targeted (that is, a representative single-nucleotide polymorphism in a genomic region with a high degree of linkage disequilibrium with groups of other polymorphic loci that make up the haplotype).

Single nucleotide polymorphism rs12566098, position chr1:67423888 (GRCh38.p14) [https://www.ncbi.nlm.nih.gov/snp/rs12566098] is localized in the intron and is characterized by the C>G,T substitution. However, the T allele occurs with a frequency of <0.000001 in European populations and is also characterized by a low frequency in other populations of the world, and therefore the C>G substitution is the most relevant for research. According to GTex Portal transcriptomic analysis, rs12566098 affects the expression level of SERBP1 [https://gtexportal.org/home/].

This creates a need to create a method for identifying the targeted polymorphic variant rs12566098 (C>G) of the SERBP1 gene that is simple to implement, inexpensive and accessible to all researchers working in the field of genetic epidemiology.

There is a known method for analyzing genetic variations in the human genome by sequencing amplified sections of DNA [Mardis E. R. DNA sequencing technologies: 2006-2016 //Nature protocols. - 2017. - Vol. 12. - No. 2. - P. 213-218]. The disadvantages of the method are the high cost of equipment and reagents, which excludes the widespread implementation of the method in experimental studies, especially the study of multifactorial diseases that require large sample sizes to ensure high power of studies.

There is a known method for analyzing genetic variations in the human genome using matrix-assisted laser desorption ionization mass spectrometry (MALDI). The method involves transferring the DNA to be analyzed onto a substrate, where it crystallizes with a matrix. The crystallized analytes are then transferred and irradiated with a laser, causing desorption and ionization of the molecules in a vacuum chamber. Positively charged DNA ions are accelerated and migrate through a vacuum tube to a highly sensitive detector at different speeds depending on the mass of the ions, resulting in different times of flight. Using the time of flight of individual ionized DNA analytes, the system determines mass and displays a mass spectrum identifying various genetic targets [Li D. et al. MALDI-TOF mass spectrometry in clinical analysis and research //ACS Measurement Science Au. - 2022. - Vol. 2. - No. 5. - P. 385-404]. The disadvantages of the method are labor intensity, high cost of equipment, high cost of the experiment, and the presence of highly qualified personnel.

The commercial genotyping kit rs12566098 (C/G) SERBP1 (Assay ID C___2694736_10; catalog number 4351379) from ThermoFisher was selected as the prototype. However, genotyping using a commercial kit is characterized by high cost, and information about the structure of primers and allele-specific probes necessary for PCR is closed to researchers, and therefore these methods cannot be reproduced in PCR laboratories with a standard set of equipment and reagents.

Thus, there is a real need to create a fast, inexpensive and easily reproducible method for identifying the rs12566098 (C>G) polymorphism of the SERBP1 gene, which could be used as a “routine” genotyping method in PCR laboratories with a standard set of equipment and reagents.

The technical result of this invention is the development of an easy-to-use and economically feasible method for genotyping the single nucleotide polymorphism rs12566098 (C>G), localized at position chr1:67423888 (GRCh38.p14) of the SERBP1 gene (Gene ID: 26135) using the polymerase chain reaction method in “real” mode time" using allele-specific signal probes containing FAM and ROX fluorophores.

The technical result is achieved by the fact that the identification of allelic variants rs12566098 (C>G) of the SERBP1 gene is carried out using the forward general primer 5′-CAGTTGTATGACAATGGTAAAATGA-3′ (SEQ ID NO 1), the reverse general primer 5′-CTCCCAAAGTGCTGGGATTA-3′ (SEQ ID NO 2), C-allele-specific fluorescently labeled probe 5′- (FAM)CACATAAA C CACTCTTTTAAG(RTQ1)-3′ (SEQ ID NO 3), G-allele-specific fluorescently labeled probe 5′- (ROX) CACATAAA G CACTCTTTTAAG(BHQ2)-3′ (SEQ ID NO 4).

The invention is illustrated by the following figure: discrimination of alleles at the rs12566098 locus of the SERBP1 gene when genotyping by real-time PCR using allele-specific fluorescent probes according to RFU values (relative fluorescence units) on a CFX96 amplifier: rs12566098-C/C genotypes are shown in orange circles, rs12566098-C/G genotypes are shown as green triangles, rs12566098-G/G genotypes are shown as blue squares; The black diamond indicates the negative control.

Work on the design of oligonucleotides included several stages:

1) Using the open database Ensembl genome browser 109 [https://www.ensembl.org/index.html], a synvenge flanking the desired single nucleotide C/G substitution rs12566098 SERBP1 was selected, and then using the available online software Primer3web version 4.1 .0 [https://primer3.ut.ee/] the sequence of oligonucleotides used to carry out the PCR reaction has been selected:

forward general primer rs12566098 5′-CAGTTGTATGACAATGGTAAAATGA-3′ (SEQ ID NO 1),

reverse general primer rs12566098 5′-CTCCCAAAGTGCTGGGATTA-3′ (SEQ ID NO 2). The size of the SERBP1 gene fragment amplified during PCR using these primers is 156 nucleotide pairs:

2) For the design of probes, we used practical recommendations [Basu C. (ed.). PCR primer design. - New york: Humana Press, 2015]. Hydrolysis probes were used in the reaction. The probe sequence was selected so that it would anneal to the template between the forward and reverse primers. Each probe was equipped with a fluorophore and a fluorescence quencher whose absorption spectrum corresponded to the wavelengths of the fluorophore spectrum. To quench FAM fluorescence, we used the RTQ1 quencher; for ROX fluorescence quenching - BHQ2 quencher. Based on the stated criteria and practical recommendations, probes with the following structure were selected:

rs12566098-C-allele-specific fluorescently labeled probe 5′- (FAM)CACATAAACCACTCTTTTAAG(RTQ1)-3′(SEQ ID NO 3),

rs12566098-G allele-specific fluorescently labeled probe 5′- (ROX)CACATAAAGCACTCTTTTAAG(BHQ2)-3′ (SEQ ID NO 4).

3) The production of primers and probes was carried out at the service center of NPK Syntol, Moscow.

4) Using practical experiments, the optimal conditions for genotyping were selected, which include the following steps: 95°C for 10 minutes, then 39 cycles [95°C for 15 seconds and 56°C for 1 minute].

5) The developed method was tested on 95 DNA samples of healthy individuals from the biobank of the Research Institute of Genetic and Molecular Epidemiology of KSMU. Genotyping was carried out according to the RFU (relative fluorescence units) values of the probe with fluorescent dyes. According to the results of genotyping rs12566098, 5 people (5.3%) turned out to be homozygous for the C allele (genotype C/C); 31 people (32.6%) were heterozygotes (genotype C/G), 59 people (62.1%) were homozygotes G/G.

6) Validation of the method was carried out with the method of mass spectrometric analysis on a genomic time-of-flight mass spectrometer MassArray analyzer 4 (Agena Bioscience). The results of both genotyping methods completely coincided (100% of genotypes). However, the patented method for genotyping the polymorphic locus rs12566098 (C>G) of the SERBP1 gene using real-time PCR using allele-specific probes allows to significantly (by 6 hours) reduce the time of analysis, and also reduces the cost of analysis (by 4-5 once).

The method is carried out as follows:

1. Isolation of DNA from peripheral venous blood. At the first stage, 0.5 ml of PBS was added to 0.5 ml of blood and centrifuged for 10 min at 12 thousand rpm. The supernatant was discarded, 1 ml of PBS was added and centrifuged again under the same conditions. The supernatant was discarded, 200 μl of TE buffer was added, pipetted until the precipitate dissolved, and then 10 μl of 1% sodium dodecyl sulfate SDS and 5 μl of proteinase K were sequentially added. The tubes were incubated in a thermostat at t=37°C for 12 hours. During the second stage Four successive centrifugations with phenol and chloroform were performed according to the protocol (10 min, 8 thousand rpm), after which the DNA was precipitated with an ice-cold solution of 95% ethyl alcohol and centrifuged for 10 min at 14.3 thousand rpm. After the alcohol had evaporated, the DNA was dissolved in 100 μl of deionized distilled water. The resulting DNA solution in water had a purity in the range A260/280=1.5-2.0 and an average concentration of about 180-200 ng/μl.

2. Preparation of DNA samples for genotyping. The quality of the isolated DNA was assessed by the degree of purity and concentration of the solution using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). All analyzed DNA samples were diluted with deionized water to a concentration of 15-20 ng/μl at A260/280=1.5-2.0.

3. Real-time polymerase chain reaction analysis of the rs12566098 (C>G) polymorphism of the SERBP1 gene using allele-specific probes. For genotyping, two flanking primers were used, forward (SEQ ID NO 1) and reverse (SEQ ID NO 2), as well as allele-specific probes: C-allele-specific fluorescently labeled probe (SEQ ID NO 3), G-allele- specific fluorescently labeled probe (SEQ ID NO 4).

Real-time PCR was carried out in 25 ml of a reaction mixture containing 1.25 U of Hot Start Taq DNA polymerase (Biolabmix, Novosibirsk, Russia), 20 ng of DNA, 10 μM of each primer, 5 μM of each probe, 0.03 mM each dNTP, 2.5 mM MgCl2; 1xPCR buffer [67 mM Tris-HCl, pH 8.8, 16.6 mM (NH4)2SO4, 0.01% Tween-20]. The amplification reaction consisted of a heating step to 95°C for 10 minutes, then 39 cycles (95°C for 15 seconds and 56°C for 1 minute).

4. Genotyping. When performing PCR in a cycler with fluorescent detection (Bio-Rad CFX96 or similar cycler), genotyping is carried out according to RFU (relative fluorescence units) values. For rs12566098 (C>G) of the SERBP1 gene, the probe with the fluorescent dye FAM corresponds to the C allele, and the probe with the ROX dye corresponds to the G allele (Fig.). The figure shows a clear division of the samples into clusters, where the black diamond corresponds to the negative control, the cluster of orange circles corresponds to the probe with the fluorescent dye FAM and allows the identification of C/C homozygotes. The cluster of blue squares corresponds to the ROX dye probe and allows the identification of G/G homozygotes. The green triangle cluster allows identification of C/G heterozygotes.

Summary

Thus, an effective and inexpensive method has been developed for the rapid identification of allelic variants C and G of the rs12566098 polymorphism of the SERBP1 gene in humans using real-time PCR using allele-specific fluorescent probes, which can be used in medicine to determine the hereditary predisposition to the development of diseases associated with carriage of the rs12566098 polymorphic variant of the SERBP1 gene, as well as for scientific purposes.

Claims (1)

A set of primers and probes for genotyping the polymorphic locus rs12566098 (C>G) of the SERBP1 gene in humans using real-time PCR, characterized in that the direct general primer 5′-CAGTTGTATGACAATGGTAAAATGA-3′ (SEQ ID NO: 1), reverse general primer 5′-CTCCCAAAGTGCTGGGATTA-3′ (SEQ ID NO: 2), and as probes – C-allele-specific fluorescently labeled probe 5′-(FAM)CACATAAACCACTCTTTTAAG(RTQ1)-3′ (SEQ ID NO: 3) and G-allele-specific fluorescently labeled probe 5′-(ROX)CACATAAAGCACTCTTTTAAG(BHQ2)-3′ (SEQ ID NO: 4).