RU2834032C1 - METHOD FOR GENOTYPING POLYMORPHIC LOCUS rs7907606 (T>G) IN HUMAN BY REAL-TIME PCR USING ALLELE-SPECIFIC FLUORESCENT PROBES - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to a method for genotyping a polymorphic locus rs7907606 (T>G) in a human by real-time PCR. Said method involves identification of allelic variants of rs7907606 (T>G), which is carried out using forward and reverse primers rs7907606 with SEQ ID NO: 1 and 2, respectively, as well as rs7907606-T- and G-allele-specific fluorescent-labelled probes with SEQ ID NO: 3 and 4, respectively.

EFFECT: present invention provides an easy-to-implement and cost-effective method of genotyping single-nucleotide polymorphism rs7907606 (T>G) by real-time polymerase chain reaction using allele-specific signalling probes containing FAM and ROX fluorophores.

1 cl, 1 dwg

Description

The 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 rs7907606 (T>G) molecular genetic research method.

State of the art.Polymorphic variant rs7907606 (T>G), localized in the intergenic regionSLK, STN1 was identified by genome-wide association studies as a variant associated with uterine fibroids and basal cell carcinoma [https://www.ebi.ac.uk/gwas/variants/rs7907606]. SNP rs7907606, position chr10:103920874 (GRCh38.p14) (https://www.ncbi.nlm.nih.gov/snp/rs7907606) is characterized by the T>G substitution. The high clinical diagnostic significance of this genetic variant regarding the risk of developing uterine fibroids and basal cell carcinoma creates a need for a simple to perform, inexpensive and accessible to researchers working in the field of genetic epidemiology, method for identifying the rs7907606 single nucleotide polymorphism (T>G).

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 are the high cost of equipment and reagents, which excludes the widespread implementation of the method in experimental studies, especially the study of diseases that require large sample sizes to ensure high research power.

A method for analyzing genetic variations in the human genome using matrix-assisted laser desorption ionization mass spectrometry (MALDI) is known. 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 the 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 the mass and displays a mass spectrum identifying different 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 need for highly qualified personnel.

The commercial genotyping kit rs7907606 (T/G) (C_189504960_10; catalog 4351379) from ThermoFisher was chosen as a prototype. However, genotyping using commercial kits is characterized by high cost, and information on the structure of the primers and allele-specific probes required 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 rapid, inexpensive and easily reproducible method for identifying the rs7907606 (T>G) polymorphism, with a primer and allele-specific probe structure available 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 rs7907606 (T>G), localized in the position chr10:103920874 (GRCh38.p14) by the polymerase chain reaction method in the "real time" mode using allele-specific signal probes containing FAM and ROX fluorophores.

The technical result is achieved in that the identification of allelic variants of rs7907606 (T>G) is carried out using the forward primer rs7907606 5′-TGCCTGAGAAAACTCAAGACTG-3′ (SEQ ID NO 1), the reverse primer rs7907606 5′-TAGGCTCCTACCCTCCAACA-3′ (SEQ ID NO 2), the rs7907606-T-allele-specific fluorescently labeled probe 5′-(FAM)CTGGAACAAACAGTAAATT A TTCTTCTTC(RTQ1)-3′ (SEQ ID NO 3), the rs7907606-G-allele-specific fluorescently labeled probe 5′-(ROX)CTGGAACAAACAGTAAATT C TTCTTCTTC(BHQ2)-3′ (SEQ ID NO 4).

The invention is explained by the following Fig. 1: discrimination of alleles at the rs7907606 locus (T>G) during genotyping by the PCR method in the "real time" mode using allele-specific fluorescent probes according to RFU (relative fluorescence units) values on a CFX96 amplifier: rs7907606-T/T genotypes are shown by orange circles, rs7907606-T/G genotypes are shown by green triangles, rs7907606-G/G genotypes are shown by 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 [T/G] rs7907606 was selected, and then, using the available online software Primer3web version 4.1.0 [https://primer3.ut.ee/], a sequence of oligonucleotides used to carry out the PCR reaction was selected:

forward common primer rs7907606 5′-TGCCTGAGAAAACTCAAGACTG-3′ (SEQ ID NO 1),

reverse common primer rs7907606 5′-TAGGCTCCTACCCTCCAACA-3′ (SEQ ID NO 2).

The size of the fragment amplified during PCR is 175 nucleotide pairs:

(TGCCTGAGAAAACTCAAGACTGCCAAAAGTAACAAACCTGCACGTTGTGCACATGTACCCTAGAACTTAAAGTATAATTAAAAAAAAAAAGAAGAAGAA [T/G] AATTTACTGTTTGTTCCAGCCAATACCTGAAGGCAAGGCCTTGTCTCCTAGCCTTTGTTGGAGG

GTAGGAGCCTA).

2) The practical recommendations [Basu C. (ed.). PCR primer design. - New York : Humana Press, 2015] were used for probe design. Hydrolysis probes were used in the reaction. The probe sequence was selected so that it annealed to the template between the forward and reverse primers. The probes were selected for the reverse DNA strand. Each probe was supplied with a fluorophore and a fluorescence quencher, the absorption spectrum of which corresponds to the wavelengths of the fluorophore spectrum. The RTQ1 quencher was used to quench FAM fluorescence; the BHQ2 quencher was used to quench ROX fluorescence.

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

rs7907606-T-allele-specific fluorescently labeled probe 5′-(FAM)CTGGAACAAACAGTAAATT A TTCTTCTTC(RTQ1)-3′ (SEQ ID NO 3), rs7907606-G-allele-specific fluorescently labeled probe 5′-(ROX)CTGGAACAAACAGTAAATT C TTCTTCTTC(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 62°C for 1 minute].

5) The developed method was tested in the laboratory of genomic studies on 200 DNA samples of healthy individuals of 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 rs7907606 genotyping, 142 individuals (71%) were homozygotes for the T allele (genotype T/T); 46 individuals (23%) were heterozygotes (genotype T/G), 12 individuals (6%) were homozygotes for the G allele (genotype G/G).

6) The method was validated by mass spectrometric analysis on a MassArray analyzer 4 genomic time-of-flight mass spectrometer (Agena Bioscience). The results of both genotyping methods were completely (100% of genotypes) identical. However, the patented method of genotyping the polymorphic locus rs7907606 (T>G) by PCR in "real time" mode using allele-specific probes allows to significantly (by 6 hours) reduce the analysis time, and also reduces the cost of the analysis (by 4-5 times).

Implementation of the invention.

The method is carried out as follows:

1. Isolation of DNA from peripheral venous blood. In the first step, 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 poured off, 1 ml of PBS was added and centrifuged again under the same conditions. The supernatant was decanted, 200 μl of TE buffer was added, the mixture was pipetted until the sediment dissolved, and then 10 μl of 1% sodium dodecyl sulfate SDS and 5 μl of proteinase K were successively added. The tubes were incubated in a thermostat at t=37°C for 12 h. During the second stage, four successive 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 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 rs7907606 (T>G) polymorphism 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: T-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, 3.5 mM MgCl2; 1x PCR 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 50°C for 2 min, 95°C for 10 min, then 39 cycles [95°C for 10 sec and 62°C for 1 min].

4. Genotyping. When performing PCR in a fluorescent detection amplifier (Bio-Rad CFX96 or similar amplifier), genotyping is performed based on RFU (relative fluorescence units) values. For rs7907606 (T>G), the probe with the fluorescent dye FAM corresponds to the T allele, and the probe with the dye ROX 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, the cluster of orange circles corresponds to the probe with the fluorescent dye FAM and allows identification of T/T homozygotes. The cluster of blue squares corresponds to the probe with the dye ROX and allows identification of G/G homozygotes. The cluster of green triangles corresponds to the accumulation of the fluorescence level for both probes and allows identification of T/G heterozygotes.

Resume.

Thus, an effective and inexpensive method for rapid identification of the polymorphic variant rs7907606 (T>G) in humans by PCR in real time 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 carriage of this polymorphism, as well as for scientific purposes.

--->

<?xml version="1.0" encoding="UTF-8"?>

<!DOCTYPE ST26SequenceListing PUBLIC "-//WIPO//DTD Sequence Listing

1.3//EN" "ST26SequenceListing_V1_3.dtd">

<ST26SequenceListing dtdVersion="V1_3" fileName="Method

genotyping of the polymorphic locus rs7907606 (T G) in humans

by the PCR method in real time using

allele-specific fluorescent probes.xml" softwareName="WIPO

Sequence" softwareVersion="2.3.0" productionDate="2024-05-03">

<ApplicationIdentification>

<IPOfficeCode>RU</IPOfficeCode>

<ApplicationNumberText></ApplicationNumberText>

<FilingDate></FilingDate>

</ApplicationIdentification>

<ApplicantFileReference>2030</ApplicantFileReference>

<ApplicantName languageCode="ru">Federal State

budgetary educational institution of higher education

&quot;Kursk State Medical University&quot;

Ministry of Health of the Russian Federation,</ApplicantName>

<ApplicantNameLatin>Kursk State Medical

University</ApplicantNameLatin>

<InventionTitle languageCode="en">Genotyping method

polymorphic locus rs7907606 (T&gt;G) in humans by PCR in

in real time using allele-specific

fluorescent probes</InventionTitle>

<SequenceTotalQuantity>4</SequenceTotalQuantity>

<SequenceData sequenceIDNumber="1">

<INSDSeq>

<INSDSeq_length>22</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..22</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q2">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>tgcctgagaaaactcaagactg</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="2">

<INSDSeq>

<INSDSeq_length>20</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..20</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q4">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>taggctcctaccctccaaca</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="3">

<INSDSeq>

<INSDSeq_length>29</INSDSeq_length>

<INSDSeq_moltype>DNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..29</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other DNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q6">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctggaacaaacagtaaattattcttcttc</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="4">

<INSDSeq>

<INSDSeq_length>29</INSDSeq_length>

<INSDSeq_moltype>RNA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..29</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>other RNA</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q8">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>ctggaacaaacagtaaattcttcttcttc</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (1)

A method for genotyping the polymorphic locus rs7907606 (T>G) in humans by the PCR method in the "real time" mode using allele-specific fluorescent probes, characterized in that the identification of allelic variants of rs7907606 (T>G) is carried out using the forward primer rs7907606 5′-TGCCTGAGAAAACTCAAGACTG-3′ (SEQ ID NO 1), the reverse primer rs7907606 5′-TAGGCTCCTACCCTCCAACA-3′ (SEQ ID NO 2), the rs7907606-T-allele-specific fluorescently labeled probe 5′-(FAM) CTGGAACAAACAGTAAATTTATTCTCTTC (RTQ1)-3′ (SEQ ID NO 3), rs7907606-G allele-specific fluorescently labeled probe 5′-(ROX)CTGGAACAAACAGTAAATT C TTCTTCTTC(BHQ2)-3′ (SEQ ID NO 4).