RU2819840C1 - METHOD FOR GENOTYPING POLYMORPHIC LOCUS rs6677 (T>G) OF C11orf58 GENE IN HUMAN BY REAL-TIME PCR USING ALLELE-SPECIFIC FLUORESCENT PROBES - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, molecular genetic diagnostics, in particular, to a method for genotyping the polymorphic locus rs6677 (T>G) of the C11orf58 gene in a human by real-time PCR using allele-specific fluorescent probes, involving PCR using specially selected primers (forward 5'-TTGTTATAGTAGAGCTGTTCATTATGG-3' and reverse 5'-TGTGAAAAGTACTTGCTCTCATGTT-3') and probes with fluorophores (T-allele-specific fluorescent-labelled probe 5'-(FAM)CTTAGTTTCCTGTTACT(RTQ1)-3' and G-allele-specific fluorescent-labelled probe 5'-(ROX)CTTAGTTGCCTGTTACT(BHQ2)-3') in an amplifier with fluorescence detection.

EFFECT: invention widens the range of methods for genotyping polymorphic variants of the C11orf58 gene, is characterized 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 rs6677 (T>G) gene C11orf58 molecular genetic research method.

State of the art

Gene C11orf58 encodes a protein discovered in 2020 by Japanese scientists [Tsuboyama K. et al. A widespread family of heat-resistant obscure (Hero) proteins protect against protein instability and aggregation //PLoS Biology. - 2020. - Vol. 18. - No. 3. - P. e3000632]. This protein is characterized by high resistance to adverse physical and chemical factors and promotes the restoration of the native tertiary or quaternary structure of other proteins under stress conditions, which proves its pronounced chaperone-like properties. The potentially significant role of this protein in a wide range of diseases (neurodegenerative, atherosclerosis, etc.) determines the need for molecular genetic studies of the C11orf58 gene and the development of methods for genotyping polymorphic variants of this gene. The C11orf58 gene (Gene ID: 10944) is located on chromosome 11p15.2. Polymorphic variant rs6677 of the C11orf58 gene, position chr11:16755585 (GRCh38.p14) [https://www.ncbi.nlm.nih.gov/snp/rs6677] is localized in the 3′-untranslated region and is characterized by a T>G substitution. According to SNPinfo Web Server/LD TAG SNP Selection [https://snpinfo.niehs.nih.gov/snpinfo/snptag.html] rs6677 of the C11orf58 gene is a target (that is, a representative single nucleotide polymorphism in a genomic region with a high degree of linkage disequilibrium with groups other polymorphic loci that make up the haplotype).

Genetic variant rs6677 (T>G) characterized by high regulatory potential. According to bioinformatics resources, this genetic variant affects the level of gene expression C11orf58through cis-eQTL effects [https://gtexportal.org/home/snp/rs6677]; is associated with histone modifications that mark enhancers [https://pubs.broadinstitute.org/mammals/haploreg/detail_v4.2.php?query=&id=rs6677]. The influence of rs6677 on binding to transcription factors depending on the carriage of the reference/alternative alleles has also been established [http://atsnp.biostat.wisc.edu/search]. This proves the high functional significance of this genetic variant and creates the need to create a method for identifying single nucleotide polymorphism rs6677 (T>G) of the gene that is simple to implement, inexpensive and accessible to researchers working in the field of genetic epidemiology C11orf58.

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.

The prototype chosen is a 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]. However, this method is characterized by high cost, since it requires the purchase of sets of spectral chips and expensive reagents for genotyping, is characterized by the high cost of equipment, requires highly qualified personnel, and therefore it cannot be reproduced with a standard set of equipment and reagents.

To date, no methods for targeted genotyping of the rs6677 (T>G) gene C11orf58 using allele-specific fluorescent probes have been developed.

Thus, there is a real need to create a fast, inexpensive and easily reproducible method for identifying the rs6677 (T>G) polymorphism of the C11orf58 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 single nucleotide polymorphism rs6677 (T>G), localized at position chr11:16755585 (GRCh38.p14) of the C11orf58 gene (Gene ID: 10944) using the polymerase chain reaction method in “real” mode time" using allele-specific signaling probes containing FAM and ROX fluorophores.

The technical result is achieved by the fact that the identification of allelic variants rs6677 (T>G) of the C11orf58 gene is carried out using the forward primer rs6677 5′-TTGTTATAGTAGAGCTGTTCATTATGG-3′ (SEQ ID NO 1), reverse primer rs6677 5′-TGTGAAAAGTACTTGCTCTCATGTT-3′ (SEQ ID NO 2), rs6677-T-allele-specific fluorescently labeled probe 5′-(FAM)CTTAGTT T CCTGTTACT(RTQ1)-3′ (SEQ ID NO 3),

rs6677-G allele-specific fluorescently labeled probe 5′-(ROX)CTTAGTT G CCTGTTACT(BHQ2)-3′ (SEQ ID NO 4).

The invention is illustrated by the following figure: discrimination of alleles at the rs6677 locus (T>G) of the C11orf58 gene when genotyping by PCR in real time using allele-specific fluorescent probes according to RFU values (relative fluorescence units) on a CFX96 amplifier: genotypes rs6677- T/T are shown as orange circles, rs6677-T/G genotypes are shown as green triangles, rs6677-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 sequence flanking the desired single nucleotide substitution [T/G] rs6677 C11orf58 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 rs6677 5′-TTGTTATAGTAGAGCTGTTCATTATGG-3′ (SEQ ID NO 1),

reverse general primer rs6677 5′-TGTGAAAAGTACTTGCTCTCATGTT-3′ (SEQ ID NO 2).

The size of the C11orf58 gene fragment amplified during PCR is 150 nucleotide pairs:

TTGTTATAGTAGAGCTGTTCATTATGGATATTTCTGCTGCCAGTCACAATCTAAATTAATTTTGGCAAAAGATTGGGTACTTAGTT [T/G] CCTGTTACTGAGTTAGCTCTACTCTTTTGGACCAAAGCAACATGAGAGCAAGTACTTTTCACA.

2). For probe design, practical recommendations were used [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:

rs6677-T-allele-specific fluorescently labeled probe 5′-(FAM)CTTAGTT T CCTGTTACT(RTQ1)-3′ (SEQ ID NO 3),

rs6677-G allele-specific fluorescently labeled probe 5′-(ROX)CTTAGTT G CCTGTTACT(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 included the following steps: 50°C for 2 minutes, 95°C for 10 minutes, then 39 cycles [95°C for 10 seconds and 48°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 rs6677, 139 people (69.5%) turned out to be homozygous for the T allele (T/T genotype); 53 people (26.5%) were heterozygotes (genotype T/G), 8 people (4%) individuals were homozygotes for the G allele (genotype G/G).

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 rs6677 (T>G) of the C11orf58 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. Real-time polymerase chain reaction analysis of the rs6677 (T>G) polymorphism of the C11orf58 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: T-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, 3.0 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 48°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 rs6677 (T>G) of the C11orf58 gene, the probe with the fluorescent dye FAM corresponds to the T 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, the cluster of orange circles corresponds to the probe with the fluorescent dye FAM and allows the identification of T/T 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 T/G heterozygotes.

Summary

Thus, an effective and inexpensive method has been developed for the rapid identification of the rs6677 (T>G) polymorphic variant of the C11orf58 gene in humans using real-time PCR using allele-specific fluorescent probes, which can be used in medicine to determine hereditary predisposition to the development of diseases associated with carriage of C11orf58 gene polymorphisms, as well as for scientific purposes.

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Claims (1)

A method for genotyping the polymorphic locus rs6677 (T>G) of the C11orf58 gene in humans using real-time PCR using allele-specific fluorescent probes, characterized in that the identification of allelic variants rs6677 (T>G) of the C11orf58 gene is carried out using a forward primer rs6677 5'-TTGTTATAGTAGAGCTGTTCATTATGG-3' (SEQ ID NO 1), reverse primer rs6677 5'-TGTGAAAAGTACTTGCTCTCATGTT-3' (SEQ ID NO 2), rs6677-T-allele-specific fluorescently labeled probe 5'-(FAM)CTTAGTT T CCTGTTACT(RTQ1)-3' (SEQ ID NO 3), rs6677-G allele-specific fluorescently labeled probe 5'-(ROX)CTTAGTT G CCTGTTACT(BHQ2)-3' (SEQ ID NO 4).