RU2850915C1 - METHOD FOR GENOTYPING POLYMORPHIC LOCUS rs6991952 (A>G) OF EBF2 GENE IN HUMANS BY REAL-TIME PCR USING ALLELE-SPECIFIC FLUORESCENT PROBES - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of molecular genetic diagnostics and can be used in medicine to determine hereditary predisposition to the development of diseases associated with carriage of the polymorphic variant rs6991952 (A>G) of the EBF2 gene. A method is proposed for genotyping the polymorphic locus rs6991952 (A>G) of the EBF2 gene in humans using real-time PCR with allele-specific fluorescent probes, which involves performing PCR using specially selected primers (forward 5′-CACCTGCTATCAATTTGACTTGC-3′ and reverse 5′-AAAATGCCCAGTCACCCCA-3′) and probes with fluorophores (A-allele-specific fluorescently labelled probe 5′-(FAM)CCTGTCCATGTATGCCAAGA(RTQ1)-3′, G-allele-specific fluorescently labelled probe 5′-(ROX)CCTGTCCATGTGTGCCAAGA(BHQ2)-3′) in an amplifier with fluorescence detection.

EFFECT: ability to expand the arsenal of methods for genotyping polymorphic variants of FOXO1, characterised by simplicity, accuracy and low cost.

1 cl, 1 dwg

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 of the rs6991952 locus (A>G) of the EBF2 gene using a molecular genetic research method.

State of the art

The EBF2 gene polymorphism rs6991952 (A>G) was identified by genome-wide association studies as a variant associated with inguinal hernias [Jorgenson E. et al. A genome-wide association study identifies four novel susceptibility loci underlying inguinal hernia //Nature communications. - 2015. - Vol. 6. - No. 1. - P. 10130]. The EBF2 gene (Gene ID: 64641) is located on chromosome 8p21.2. Single nucleotide polymorphism rs6991952, position chr8:25849896 (GRCh38.p14) [https://www.ncbi.nlm.nih.gov/snp/rs6991952] is localized in an intron and is characterized by the A>G substitution.

The high clinical diagnostic significance of this genetic variant in relation to the risk of developing inguinal hernia creates a need for a method for identifying the single nucleotide polymorphism rs6991952 (A>G) of the EBF2 gene that is simple to implement, inexpensive, and accessible to all researchers working in the field of genetic epidemiology.

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

The prototype method chosen for analyzing genetic variations in the human genome is matrix-assisted laser desorption ionization mass spectrometry (MALDI). The method involves transferring the DNA to be analyzed onto a substrate, where it crystallizes with the 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, which results in different times of flight. Using the time of flight of individual ionized DNA analytes, the system determines the 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]. However, this method is characterized by high cost, since it requires the purchase of spectral chip sets and expensive reagents for genotyping, is characterized by high cost of equipment, requires highly qualified personnel, and therefore cannot be reproduced with a standard set of equipment and reagents.

Methods for targeted genotyping of the rs6991952 (A>G) polymorphism of the EBF2 gene using allele-specific fluorescent probes have not been developed to date.

Thus, there is a real need to create a rapid, inexpensive and easily reproducible method for identifying the rs6991952 (A>G) polymorphism of the EBF2 gene, with a structure of primers and allele-specific probes accessible to all researchers, which could be used as a “routine” genotyping method in any PCR laboratory.

Disclosure of the essence of the invention

The technical result of this invention is the development of a simple to implement and economically feasible method for genotyping the single nucleotide polymorphism rs6991952 (A>G), localized at position chr8:25849896 (GRCh38.p14) of the EBF2 gene (Gene ID: 64641) by the polymerase chain reaction method in real time using allele-specific signal probes containing FAM and ROX fluorophores.

The technical result is achieved in that the identification of allelic variants of rs6991952 (A>G) of the EBF2 gene is carried out using the forward primer rs6991952 5′-CACCTGCTATCAATTTGACTTGC-3′ (SEQ ID NO 1), the reverse primer rs6991952 5′-AAAATGCCCAGTCACCCCA-3′ (SEQ ID NO 2), rs6991952-A-allele-specific fluorescently labeled probe 5′-(FAM)CCTGTCCATGT A TGCCAAGA(RTQ1)-3′ (SEQ ID NO 3), rs6991952-G-allele-specific fluorescently labeled probe 5′-(ROX)CCTGTCCATGT G TGCCAAGA(BHQ2)-3′ (SEQ ID NO 4).

The invention is illustrated by the following figure: discrimination of alleles at the rs6991952 (A>G) locus of the EBF2 gene during genotyping by the PCR method in real time using allele-specific fluorescent probes based on RFU (relative fluorescence units) values on a CFX96 amplifier: rs6991952-A/A genotypes are shown as orange circles, rs6991952-A/G genotypes are shown as green triangles, rs6991952-G/G genotypes are shown as blue squares; the negative control is marked with a black diamond.

The 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 sequence flanking the desired single nucleotide substitution [A/G] rs6991952 of the EBF2 gene 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 was selected:

forward common primer rs6991952 5′-CACCTGCTATCAATTTGACTTGC-3′ (SEQ ID NO 1),

reverse common primer rs6991952 5′-AAAATGCCCAGTCACCCCA-3′ (SEQ ID NO 2).

The size of the DNA fragment amplified during PCR is 158 nucleotide pairs:

(CACCTGCTATCAATTTGACTTGCATGTAACCAGAATTCTCAGAACTTGTTGGTTGCTCCTGCTGGCTCCTGTCCATGT [A/G] TGCCAAGAGGACATGATGGCAAGCGGATTAGGTAAGCAGAAAGCACCATGTGGTATCCCCTGGGGTGACTGGGCATTTT).

2) For probe design, we used the 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 such that it annealed to the template between the forward and reverse primers. Each probe was equipped with a fluorophore and a fluorescence quencher, the absorption spectrum of which corresponded to the wavelengths of the fluorophore spectrum. To quench FAM fluorescence, we used the RTQ1 quencher; to quench ROX fluorescence, we used the BHQ2 quencher.

Based on the stated criteria and practical recommendations, probes with the following structure were selected:

rs6991952-A-allele-specific fluorescently labeled probe 5′-(FAM)CCTGTCCATGT A TGCCAAGA(RTQ1)-3′ (SEQ ID NO 3), rs6991952-G-allele-specific fluorescently labeled probe 5′-(ROX)CCTGTCCATGT G TGCCAAGA(BHQ2)-3′ (SEQ ID NO 4).

3) The production of primers and probes was carried out at the service center of NPK Sintol, 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 59°C for 1 minute].

5) The developed method was tested in the genomic research laboratory on 200 DNA samples of healthy individuals from the biobank of the Research Institute of Genetic and Molecular Epidemiology of KSMU. Genotyping was performed based on the RFU (relative fluorescence units) values of probes with fluorescent dyes. According to the results of genotyping of rs6991952 of the EBF2 gene, 67 individuals (33.5%) were homozygotes for the A allele (genotype A/A); 101 individuals (50.5%) were heterozygotes (genotype A/G); 32 individuals (16.0%) were homozygotes for the G allele (genotype G/G).

6) The method was validated using mass spectrometric analysis on a MassArray analyzer 4 genomic time-of-flight mass spectrometer (Agena Bioscience). The results of both genotyping methods were completely consistent (100% of genotypes). However, the patented method for genotyping the rs6991952 (A>G) polymorphic locus of the EBF2 gene using real-time PCR with allele-specific probes significantly reduces analysis time (by 6 hours) and also reduces the cost of analysis (by 4-5 times).

Implementation of the invention

The method is carried out as follows:

1. DNA extraction from peripheral venous blood. In the first step, 0.5 ml of PBS was added to 0.5 ml of blood and the sample was centrifuged for 10 min at 12,000 rpm. The supernatant was decanted, 1 ml of PBS was added, and the sample was centrifuged again under the same conditions. The supernatant was decanted, 200 μl of TE buffer was added, the mixture was pipetted until the precipitate dissolved, and then 10 μl of 1% sodium dodecyl sulfate (SDS) solution and 5 μl of proteinase K were added sequentially. The tubes were incubated in a thermostat at t = 37 °C for 12 h. During the second stage, four sequential centrifugations with phenol and chloroform were carried out according to the protocol of the method (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 evaporation of the alcohol, the DNA was dissolved in 100 μl of deionized distilled water. The resulting DNA solution in water had a purity in the range of 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 based on purity and solution concentration 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 an A260/280 ratio of 1.5-2.0.

3. Analysis of the rs6991952 (A>G) polymorphism of the EBF2 gene by 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: A-allele-specific fluorescently labeled probe (SEQ ID NO 3), G-allele-specific fluorescently labeled probe (SEQ ID NO 4).

Real-time PCR was performed in a 25 ml reaction mixture containing 1.25 U Hot Start Taq DNA polymerase (Biolabmix, Novosibirsk, Russia), 20 ng DNA, 10 μM of each primer, 5 μM of each probe, 0.03 mM of 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 min, 95°C for 10 min, then 39 cycles [95°C for 10 sec and 59°C for 1 min].

4. Genotyping. When performing PCR in a thermocycler with fluorescence detection (Bio-Rad CFX96 or a similar thermocycler), genotyping is performed based on the RFU (relative fluorescence units) values. For rs6991952 (A>G) of the EBF2 gene, the probe with the fluorescent dye FAM corresponds to the A allele, and the probe with the ROX dye corresponds to the G allele (Fig. 1). The figure shows a clear division of the samples into clusters, where the black diamond corresponds to the negative control, and the cluster of orange circles corresponds to the probe with the fluorescent dye FAM and allows for the identification of A/A homozygotes. The cluster of green triangles corresponds to the accumulation of fluorescence levels for both probes and allows for the identification of A/G heterozygotes. The cluster of blue squares corresponds to a probe with the fluorescent dye ROX and allows identification of G/G homozygotes.

Resume

Thus, an effective and inexpensive method for rapid identification of the polymorphic variant rs6991952 (A>G) of the EBF2 gene in humans by real-time PCR using allele-specific fluorescent probes has been developed, which can be used in medicine to determine the predisposition to the development of diseases associated with the carriage of EBF2 gene polymorphisms, as well as for scientific purposes.

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Claims (1)

A method for genotyping the polymorphic locus rs6991952 (A>G) of the EBF2 gene in humans by PCR in real time using allele-specific fluorescent probes, characterized in that the identification of allelic variants of rs6991952 (A>G) of the EBF2 gene is carried out using the forward primer rs6991952 5′-CACCTGCTATCAATTTGACTTGC-3′ (SEQ ID NO 1), the reverse primer rs6991952 5′-AAAATGCCCAGTCACCCCA-3′ (SEQ ID NO 2), the rs6991952-A-allele-specific fluorescently labeled probe 5′-(FAM)CCTGTCCATGT A TGCCAAGA(RTQ1)-3′ (SEQ ID NO 3), rs6991952-G allele-specific fluorescently labeled probe 5′-(ROX)CCTGTCCATGT G TGCCAAGA(BHQ2)-3′ (SEQ ID NO 4).