FIELD: biotechnology.
SUBSTANCE: method for detection of expansion of trinucleotide CGG repeats in 5'-untranslated, promoter region of the FMR1 gene in case of ataxia/tremor syndrome associated with fragile X-chromosome (FXTAS), which is performed by DNA sample examination by polymerase chain reaction (PCR). The length of the amplified fragments was analyzed by capillary gel electrophoresis with laser-induced fluorescent detection of polymerase chain reaction products with a fluorescently labeled primer and subsequent detection of expansion of CGG repeats in the DNA sample associated with FXTAS. At that, detection of the presence of CGG repeats expansion in the DNA sample is performed by amplifying the locus of tandem CGG repeats of the FMR1 gene promoter region in PCR using two original specific oligonucleotide primers: direct FMR1 F: 5'-CGCTCAGCTCCGTTTCGG-3' and reverse, labeled with a fluorescent label - FAM, FMR1 R: 5'-(FAM)AAGTACCTTGTAGAAAGCGCCA-3', flanking the analyzed region of DNA. Moreover, the PCR reaction of DNA fragments containing tandem CGG repeats is carried out in a reaction medium containing: 50 mM of KCl, 50 mM of Tris-HCl with a pH of 8.8, 2.5 mM of MgCl2, 250 mcM of deoxyribonucleotide triphosphate (dNTP), 1M of betaine, 100 mcM' of 7-diazaguanazine-5 -triphosphate (7-deaza-GTP), 5% dimethylsulfoxide (DMSO), 0.5 units of thermostable DNA polymerase with antibodies inhibiting the enzyme activity, and 0.5 pmol of each primer, direct and reverse, under the following conditions: initial DNA melting is carried out for 900 seconds at 98°C. Next, 35 amplification cycles involving denaturation at 97°C for 45 seconds, primers annealing at a temperature of 62°C for 30 seconds and DNA portion synthesis at a temperature of 72°C for 240 seconds. And the final elongation is carried out at a temperature of 72°C for 1500 seconds, and then the size of the obtained fluorescently-labeled DNA amplicon fragments is estimated by fragment analysis on a capillary genetic sequencer. For the detected electrophoregram peaks, the number of CGG repeats in the promoter region of the FMR1 (N) gene is calculated by the formula: N=(1.04X-237)/3, where 1.04 is the correction factor; X is the size of the amplicon of the DNA sample of the corresponding electrophoregram peak; 237 is the size of the amplicon region outside the repeats (5'-end and 3'-end); 3 is the number of nucleotides in one repeat. In case of detection of a single peak in the electrophoregram for women, and absence of a detectable peak for men, the presence of CGG repeats expansion in the DNA sample is determined repeatedly by amplifying the locus of tandem CGG repeats of the FMR1 gene promoter region in PCR using the same oligonucleotide primers: direct FMR1 F: 5'-CGCTCAGCTCCGTTTCGG-3' and reverse FMR1 R: 5'-AAGTACCTTGTAGAAAGCGCCA-3' without label, followed by obtained amplicons separation by electrophoresis. And the number of CGG repeats in the FMR1 gene promoter region (N) is calculated by the formula: N=(X-237)/3, where X is the size of the DNA sample amplicon of the corresponding electrophoregram strip; 237 is the amplicon area out of repeats (5'-end to 3'-end); 3 is the number of nucleotides in one repeat. The invention can be used to identify mikrosatellite genotyping of tandem-CGG repeats expansion in the FMR1 gene (fragile X mental retardation 1) in case of FXTAS (Fragile X-associated tremor/ataxia syndrome tremor/ataxia syndrome; associated with fragile X chromosome) disease.
EFFECT: increased accuracy of expansion detection due to the high sensitivity of the method.
2 cl, 4 dwg, 1 tbl
Title | Year | Author | Number |
---|---|---|---|
PREIMPLANTATION METHOD OF MARTINE-BELL SYNDROME GENETIC TESTING | 2022 |
|
RU2796834C1 |
LARGE-SCALE GENOTYPING OF DISEASES AND DIAGNOSTIC TEST FOR CEREBELLAR ATAXIA OF TYPE 6 | 1998 |
|
RU2195497C2 |
SYNTHETIC OLIGONUCLEOTIDE PRIMERS AND METHOD FOR GENETIC TYPING FOR PERSONAL IDENTIFICATION BY Y-CHROMOSOME MICROSATELLITE DNA MARKERS SYSTEM | 2012 |
|
RU2528742C2 |
METHOD OF DETERMINATION OF HAPLOTYPIC POLYMORPHISM OF INDIVIDUAL AUTOSOMAL DNA SITE | 2010 |
|
RU2432398C1 |
SEQUENCE GRAPH-BASED TOOL FOR DETERMINING VARIATION IN SHORT TANDEM REPEAT AREAS | 2020 |
|
RU2799654C2 |
SEQUENCE GRAPH TOOL FOR DETERMINING VARIATIONS IN REGIONS OF SHORT TANDEM REPEATS | 2020 |
|
RU2825664C2 |
TEST SYSTEM FOR PRE-IMPLANTATION GENETIC TESTING OF TYPE 1 SPINOCEREBELLAR ATAXIA | 2021 |
|
RU2769300C1 |
SET OF STR-MARKERS OF THE Y-CHROMOSOME FOR DETERMINING THE ETHNO-TERRITORIAL ORIGIN OF AN INDIVIDUAL BASED ON A SAMPLE OF HIS DNA | 2021 |
|
RU2804433C2 |
METHOD OF GENETIC TYPING OF YERSINIA PESTIS AND YERSINIA PSEUDOTUBERCULOSIS BASED ON THE ANALYSIS OF 13 VNTR LOCUSES IN MULTIPLEX PCR REACTION | 2017 |
|
RU2644236C1 |
METHOD OF SIMULTANEOUS DETERMINATION OF PERSON'S GENOTYPE BASED ON POLYMORPHISMS IN THREE GENES INVOLVED IN REGULATION OF BEHAVIOR | 2021 |
|
RU2808826C2 |
Authors
Dates
2017-05-30—Published
2016-12-27—Filed