RU2821925C1 - METHOD FOR GENOTYPING POLYMORPHIC LOCUS rs67579710 (G>A) OF THBS3 GENE IN HUMAN BY REAL-TIME PCR USING ALLELE-SPECIFIC FLUORESCENT PROBES - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology, in particular to a method for genotyping a polymorphic locus rs67579710 (G>A) of the THBS3 gene in a human by real-time PCR using primers SEQ ID NO: 1, SEQ ID NO: 2 and allele-specific fluorescent probes SEQ ID NO: 3, SEQ ID NO: 4 in an amplifier with fluorescence detection.

EFFECT: invention is effective for accurate and cheap genotyping of human THBS3 gene polymorphic locus rs67579710 (G>A).

1 cl, 1 dwg, 1 ex

Description

Field of technology to which the invention relates

The invention relates to the field of biotechnology, molecular genetic diagnostics, in particular to the assessment of single nucleotide polymorphism rs67579710 (G>A) of the THBS3 gene using a molecular genetic research method.

State of the art

The polymorphic marker rs67579710 (G>A) of the THBS3 gene was identified by genome-wide association studies as a variant associated with severe COVID-19 [https://www.ebi.ac.uk/gwas/variants/rs67579710]. The THBS3 gene (Gene ID: 7059) is located on chromosome 1q22. Single nucleotide polymorphism rs67579710, position chr1:155203736 (GRCh38.p14) (https://www.ncbi.nlm.nih.gov/snp/rs67579710) is localized in the intron and is characterized by the G>A substitution.

The high clinical and diagnostic significance of this genetic variant in the relatively severe course of COVID-19 creates the need to create a method for identifying the single nucleotide polymorphism rs67579710 (G>A) of the THBS3 gene that is simple to implement, inexpensive and accessible to researchers working in the field of genetic epidemiology.

There is a known method for analyzing genetic variations in the human genome by sequencing amplified DNA sections [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 precludes the widespread implementation of the method in experimental studies, especially the study of diseases that require large sample sizes to ensure high power of research.

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 rs67579710 (G/A) THBS3 (C__96698905_10; catalog 4351379) from ThermoFisher was selected as the prototype. However, genotyping using commercial kits 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 it cannot be reproduced 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 rs67579710 (G>A) polymorphism of the THBS3 gene, with a structure of primers and allele-specific probes available to all researchers, which could be used as a “routine” genotyping method in any PCR laboratory.

Disclosure of the invention

The technical result of this invention is the development of an easy-to-use and economically feasible method for genotyping the single nucleotide polymorphism rs67579710 (G>A), localized at position chr1:155203736 (GRCh38.p14) of the THBS3 gene (Gene ID: 7059) 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 rs67579710 (G>A) of the THBS3 gene is carried out using the forward primer rs67579710 5'- AATAAAGATGGGGTGCTGGA-3' (SEQ ID NO 1), the reverse primer rs67579710 5'-TCCATCCCTTCTGCCACTAC-3' (SEQ ID NO 2), rs67579710-G-allele-specific fluorescently labeled probe 5'-(FAM)AGGCCCTCTAAGGGAGTTGCTTCACA(RTQ1)-3'(SEQ ID NO 3), rs67579710-A-allele-specific fluorescently labeled probe 5'-( ROX)AGGCCCTCTAAGAGAGTTGCTTCACA(BHQ2)-3' (SEQ ID NO 4).

The invention is illustrated by the following drawing: discrimination of alleles at the rs67579710 locus of the THBS3 gene when genotyping by real-time PCR using allele-specific fluorescent probes according to RFU values (relative fluorescence units) on a CFX96 amplifier: rs67579710-G/G genotypes are shown in orange circles, rs67579710-G/A genotypes are shown as green triangles, rs67579710-A/A 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 substitution [G/A] rs67579710 of the THBS3 gene was selected, and then using 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 rs67579710 5'- AATAAAGATGGGGTGCTGGA-3' (SEQ ID NO 1),

reverse general primer rs67579710 5'-TCCATCCCTTCTGCCACTAC-3' (SEQ ID NO 2).

The size of the THBS3 gene fragment amplified during PCR is 159 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. The probes were selected for the reverse strand of DNA. 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:

rs67579710-G-allele-specific fluorescently labeled probe

5'-(FAM)AGGCCCTCTAAGGGAGTTGCTTCACA(RTQ1)-3'(SEQ ID NO 3),

rs67579710-A-allele-specific fluorescently labeled probe

5'-(ROX)AGGCCCTCTAAGAGAGTTGCTTCACA(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, optimal conditions for genotyping were selected, which include the following steps: 50°C for 2 minutes, 95°C for 10 minutes, then 39 cycles [95°C for 10 seconds and 66°C for for 1 minute].

5) The developed method was tested in the laboratory of genomic research on 200 DNA samples of healthy individuals in 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 probes with fluorescent dyes. According to the results of genotyping rs67579710, 163 people (81.5%) were homozygous for the G allele (genotype G/G); 35 people (17.5%) were heterozygotes (genotype G/A), 2 people (1%) individuals were homozygous for allele A (genotype A/A).

6) Validation of the method was carried out by 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 rs67579710 (G>A) of the THBS3 gene using real-time PCR using allele-specific probes can significantly (by 6 hours) reduce the time of analysis, and also reduces the cost of analysis (by 4-5 once).

Carrying out the invention

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 sediment 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. Analysis of the rs67579710 (G>A) polymorphism of the THBS3 gene using real-time polymerase chain reaction 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: G-allele-specific fluorescently labeled probe (SEQ ID NO 3), A-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 of 50°C for 2 minutes, 95°C for 10 minutes, then 39 cycles [95°C for 10 seconds and 66°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 rs67579710 (G>A) of the THBS3 gene, the probe with the fluorescent dye FAM corresponds to the G allele, and the probe with the ROX dye corresponds to the A allele (Fig. 1). The drawing shows a clear division of 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 G/G homozygotes. The cluster of blue squares corresponds to the ROX dye probe and allows the identification of A/A homozygotes. The cluster of green triangles corresponds to the accumulation of fluorescence levels for both probes and allows the identification of G/A heterozygotes.

Summary

Thus, an effective and inexpensive method has been developed for the rapid identification of the rs67579710 (G>A) polymorphic variant of the THBS3 gene in humans using real-time PCR using allele-specific fluorescent probes, which can be used in medicine to determine susceptibility to the development of diseases associated with carriage of THBS3 gene polymorphisms, as well as for scientific purposes.

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Claims (1)

A method for genotyping the polymorphic locus rs67579710 (G>A) of the THBS3 gene in humans using real-time PCR using allele-specific fluorescent probes, characterized in that the identification of allelic variants rs67579710 (G>A) of the THBS3 gene is carried out using a forward primer rs67579710 5'-AATAAAGATGGGGTGCTGGA-3' (SEQ ID NO 1), reverse primer rs67579710 5'-TCCATCCCTTCTGCCACTAC-3' (SEQ ID NO 2), rs67579710-G-allele-specific fluorescent-labeled probe 5'-(FAM)AGGCCCTCTAAG G GAGTTGCTTCACA(RTQ1)-3' (SEQ ID NO 3), rs67579710-A allele-specific fluorescently labeled probe 5'-(ROX)AGGCCCTCTAAG A GAGTTGCTTCACA(BHQ2)-3' (SEQ ID NO 4).