CN114736960B - Molecular marker and kit for predicting sudden cardiac death based on METTL gene polymorphism sites - Google Patents
Molecular marker and kit for predicting sudden cardiac death based on METTL gene polymorphism sites Download PDFInfo
- Publication number
- CN114736960B CN114736960B CN202210521337.1A CN202210521337A CN114736960B CN 114736960 B CN114736960 B CN 114736960B CN 202210521337 A CN202210521337 A CN 202210521337A CN 114736960 B CN114736960 B CN 114736960B
- Authority
- CN
- China
- Prior art keywords
- mettl
- sudden cardiac
- cardiac death
- gene
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 206010049418 Sudden Cardiac Death Diseases 0.000 title claims abstract description 53
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 39
- 239000003147 molecular marker Substances 0.000 title abstract description 9
- 238000001514 detection method Methods 0.000 claims abstract description 11
- 230000000692 anti-sense effect Effects 0.000 claims description 9
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 238000005251 capillar electrophoresis Methods 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 5
- 238000003780 insertion Methods 0.000 abstract description 16
- 230000037431 insertion Effects 0.000 abstract description 16
- 238000012217 deletion Methods 0.000 description 15
- 230000037430 deletion Effects 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 9
- 238000011160 research Methods 0.000 description 6
- 238000011161 development Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 108020005345 3' Untranslated Regions Proteins 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 108700028369 Alleles Proteins 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000007850 fluorescent dye Substances 0.000 description 3
- 230000011987 methylation Effects 0.000 description 3
- 238000007069 methylation reaction Methods 0.000 description 3
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 206010071602 Genetic polymorphism Diseases 0.000 description 2
- 101000990485 Homo sapiens RNA N6-adenosine-methyltransferase METTL16 Proteins 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 108700040121 Protein Methyltransferases Proteins 0.000 description 2
- 102000055027 Protein Methyltransferases Human genes 0.000 description 2
- 102100030522 RNA N6-adenosine-methyltransferase METTL16 Human genes 0.000 description 2
- 206010042434 Sudden death Diseases 0.000 description 2
- 108010006785 Taq Polymerase Proteins 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000000747 cardiac effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000009456 molecular mechanism Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 206010007572 Cardiac hypertrophy Diseases 0.000 description 1
- 208000006029 Cardiomegaly Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108091033380 Coding strand Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 101100214180 Drosophila melanogaster Ythdf gene Proteins 0.000 description 1
- 208000010496 Heart Arrest Diseases 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- 101000967135 Homo sapiens N6-adenosine-methyltransferase catalytic subunit Proteins 0.000 description 1
- 101000959153 Homo sapiens RNA demethylase ALKBH5 Proteins 0.000 description 1
- 108091007767 MALAT1 Proteins 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 102000016397 Methyltransferase Human genes 0.000 description 1
- VQAYFKKCNSOZKM-IOSLPCCCSA-N N(6)-methyladenosine Chemical compound C1=NC=2C(NC)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O VQAYFKKCNSOZKM-IOSLPCCCSA-N 0.000 description 1
- 102100040619 N6-adenosine-methyltransferase catalytic subunit Human genes 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 206010064911 Pulmonary arterial hypertension Diseases 0.000 description 1
- 102100039083 RNA demethylase ALKBH5 Human genes 0.000 description 1
- 230000026279 RNA modification Effects 0.000 description 1
- 206010038669 Respiratory arrest Diseases 0.000 description 1
- 208000003443 Unconsciousness Diseases 0.000 description 1
- 208000005475 Vascular calcification Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 208000007474 aortic aneurysm Diseases 0.000 description 1
- 206010003119 arrhythmia Diseases 0.000 description 1
- 230000003143 atherosclerotic effect Effects 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 238000010562 histological examination Methods 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 208000010125 myocardial infarction Diseases 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000014493 regulation of gene expression Effects 0.000 description 1
- 230000008960 regulation of mRNA stability Effects 0.000 description 1
- 238000012502 risk assessment Methods 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a molecular marker and a kit for predicting sudden cardiac death based on METTL gene polymorphism sites, wherein the molecular marker is the rs58928048 site of METTL gene, the site is inserted or deleted with a base TGTCTG, and the insertion type indicates that the risk of sudden cardiac death is high. The invention provides a polymorphism molecular marker related to sudden cardiac death through researching the correlation between the indemnity polymorphism of the METTL gene rs58928048 locus and the sudden cardiac death phenotype, and further provides a sudden cardiac death detection kit, so that the risk of sudden cardiac death of an individual is detected.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a molecular marker and a kit for predicting sudden cardiac death based on METTL gene polymorphism sites.
Background
Sudden Cardiac Death (SCD) refers to sudden unexpected death caused by cardiac causes, manifested by sudden loss of consciousness and circulatory and respiratory arrest occurring in a short period of time. "short term" is defined as less than 1 hour with witness and 24 hours without witness. Numerous studies have previously shown that the cause of death of SCD is mostly coronary atherosclerotic heart disease or fatal cardiac arrhythmias. The former is mostly seen in the elderly population, while the latter is mostly seen in the young population. In view of post mortem autopsy and histological examination, it is difficult to find an accurate cause of death, and in order to accurately diagnose SCD, it is urgent to reveal the molecular mechanism by which SCD occurs and find genetic markers of SCD.
In the past, research in the field of SCD diagnostics has focused mainly on analysis of the association of mutations in the coding region of important protein-encoding genes with sudden death, however, the number of mutations involved is limited, and the extremely low gene frequency of the general population makes it difficult to fully interpret the relatively high incidence of sudden death. With the development of whole genome association studies and second generation sequencing technologies, more and more genetic markers are found to be involved in the regulation of SCD. The identification of the SCD genetic markers can further understand the potential mechanism, optimize the risk stratification of the SCD, and provide powerful theoretical basis for molecular diagnosis and prevention. Considering that the 3'UTR plays an important role in regulating gene expression, people focus on the genetic polymorphism in the region, and the correlation between the genetic polymorphism of the 3' UTR in related genes and the susceptibility of SCD is analyzed through case contrast research, so that the possibility is provided for establishing a gene polymorphism detection system for SCD risk assessment.
N6-methyladenosine (m 6A) is the most common and abundant type of internal post-transcriptional RNA modification in eukaryotic cells, and is reversibly and dynamically regulated by its effector proteins methyltransferases (METTL, METTL3, METTL, etc.), demethylases (FTO, ALKBH5, etc.), and related binding proteins (YTHDF 1-3). m6A plays an important role in regulation of gene expression, mRNA stability and homeostasis, various diseases, and the like. With the development of scientific technology, more and more researches find that m6A related protein glycosylation abnormality is a sign of the development of cardiovascular diseases, including cardiac hypertrophy, heart failure, myocardial infarction, ischemic heart disease, aortic aneurysm, vascular calcification, pulmonary arterial hypertension and the like. A number of m6A-SNPs associated with cardiovascular disease have also been identified using whole genome association studies (GWAS) and the mechanism of genetic function has been explored. If rs9847953 and rs197922 are identified as potential functional variations, they are helpful for regulating blood pressure. Studies have also shown that rs12286 may affect m6A methylation levels mechanically and are significantly associated with SCD. Overall, m6A methylation regulation is critical to maintaining cardiac physiological function, and m6A modification may help to further understand the molecular mechanisms of cardiovascular pathogenesis and may be a potential biomarker in the future.
METTL16 is a novel m6A methyltransferase, comprising the Rossmann-like folding of class I methyltransferases, involved in m6A modification by modulating SAM synthase or other factors. Studies have shown that METTL16 plays an important role in the development and progression of tumors (e.g., liver cancer) as a oncogene. MetTLL16 low expression has also been established in Ovarian Cancer (OC), and METTL-mediated methylation of RNA structures is an important participant in the development of mouse embryos, and further guesses suggest that MetT/Malat 1 interactions may be involved in angiogenesis.
In the prior art, no research report on the correlation between rs58928048 indel polymorphism and SCD exists, and no relevant report on the prediction of the susceptibility of SCD by detecting the insertion indel polymorphic site of 3' UTR of METTL gene exists.
Disclosure of Invention
In order to solve the technical problems, the invention provides a polymorphic molecular marker related to sudden cardiac death through researching the correlation between the insertion deletion polymorphism of the rs58928048 locus of METTL gene and the sudden cardiac death phenotype, and further provides a sudden cardiac death detection kit, so that the risk of sudden cardiac death of an individual is detected.
The first object of the present invention is to provide an indel polymorphism molecular marker for detecting risk of Sudden Cardiac Death (SCD) onset, which is the rs58928048 locus of METTL gene, which has a 6-base indel polymorphism, specifically, an indel or a deletion base TGTCTG.
Furthermore, insertion base TGTCTG (insertion type) indicates a high risk of sudden cardiac death, and deletion base TGTCTG (deletion type) indicates a low risk of sudden cardiac death.
The second object of the invention is to provide an application of METTL gene indel polymorphism site in preparing sudden cardiac death detection kit, which is used for detecting genotype of METTL gene rs58928048 site.
Further, the kit for detecting sudden cardiac death comprises a primer pair for amplifying METTL gene rs58928048 locus.
Further, the primer pair for amplifying METTL gene rs58928048 locus comprises a sense primer and an antisense primer, the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, the nucleotide sequence of the antisense primer is shown as SEQ ID NO.2, and specific sequence information is as follows:
SEQ ID NO.1：5′-CACATGAATGCTTGGACTAGAAT-3′，
SEQ ID NO.2：5′-GTGTATTTTTTTTCCTGGAGAGA-3′。
Further, for ease of identification, the primer pair is provided with an identification label, such as a fluorescent label.
The primer pair is designed for the rs58928048 insertion deletion site on METTL gene, can specifically amplify fragments containing the insertion deletion, marks fluorescent dye at the 5' end of the oligonucleotide primer by fluorescent marking technology, and one chain of the PCR amplified product carries the fluorescent dye of the marked primer. It will be appreciated by those skilled in the art that specific primer pairs can be synthesized using conventional synthetic techniques, and that the primers of the present invention are not limited to the pair of primers SEQ ID NO.1 and SEQ ID NO. 2.
Further, the Tm value of the sense primer was 57℃and the Tm value of the antisense primer was 59 ℃.
Further, the sudden cardiac death detection kit also comprises components for PCR amplification and capillary electrophoresis.
Further, the components for PCR amplification and capillary electrophoresis include Taq DNA polymerase, dNTP mixture, mgCl 2 solution, PCR reaction buffer and deionized water. The amplified products of the PCR are analyzed for the genotype of the site by capillary electrophoresis separation techniques.
Further, the preservation temperature of the kit is-20 ℃.
The principle of the invention is as follows: the inventor discovers through molecular biological research that different isogenotypes of one indel polymorphism rs58928048 in the 3' UTR of METTL gene can influence the stability of METTL gene mRNA by influencing the structure of the mRNA and possibly interact with promoters of adjacent genes as regulatory elements to remotely regulate the transcriptional activity of the adjacent genes; furthermore, it was found from SCD case-control studies in a population of a certain scale that the deletion allele of the insertion deletion polymorphism was positively correlated with the occurrence of SCD, and that this correlation was still present even after adjustment of age, sex, etc. This indel polymorphism is therefore considered useful for assessing the susceptibility of an individual to SCD.
The third purpose of the invention is to provide an application of a primer for amplifying METTL gene rs58928048 locus in preparation of a sudden cardiac death detection kit.
Further, the primer for amplifying METTL gene rs58928048 locus comprises a sense primer and an antisense primer, the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, the nucleotide sequence of the antisense primer is shown as SEQ ID NO.2, and specific sequence information is as follows:
SEQ ID NO.1：5′-CACATGAATGCTTGGACTAGAAT-3′，
SEQ ID NO.2：5′-GTGTATTTTTTTTCCTGGAGAGA-3′。
The fourth object of the invention is to provide a kit for detecting sudden cardiac death based on METTL gene indel polymorphism, which is used for detecting the genotype of the site rs58928048 of the METTL gene and comprises a primer for amplifying the site rs58928048 of the METTL gene; the primer comprises a sense primer and an antisense primer, the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, the nucleotide sequence of the antisense primer is shown as SEQ ID NO.2, and specific sequence information is as follows:
SEQ ID NO.1：5′-CACATGAATGCTTGGACTAGAAT-3′，
SEQ ID NO.2：5′-GTGTATTTTTTTTCCTGGAGAGA-3′。
By means of the scheme, the invention has at least the following advantages:
The specific primer pair contained in the kit provided by the invention is designed aiming at the rs58928048 indel site on METTL gene, can specifically amplify DNA fragments containing the site, and can identify different genotypes by detecting the mobility of fragments with different lengths in capillary electrophoresis, and according to the research of our case contrast, the rs58928048 indel site on METTL gene of detected DNA can be considered as SCD susceptibility. Therefore, the susceptibility of the individual SCD can be predicted by detecting the genotype of the rs58928048 indel site on the METTL gene of the individual.
The foregoing description is only an overview of the present invention, and is presented in terms of preferred embodiments of the present invention and the following detailed description of the invention in conjunction with the accompanying drawings.
Drawings
In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings.
FIG. 1 is a diagram showing gene sequencing and SDS-PAGE gel electrophoresis typing.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
Example 1 use of detection kit
Step 1: extraction of DNA template
Genomic DNA of peripheral blood was extracted using a blood genomic DNA extraction system (non-centrifugal column).
Step 2: PCR reaction-replication of the fragment of interest
A PCR detection kit was used that can detect SCD susceptibility, containing the following primers:
SEQ ID NO.1:5'-CACATGAATGCTTGGACTAGAAT-3', tm value is 57 ℃;
SEQ ID NO.2:5'-GTGTATTTTTTTTCCTGGAGAGA-3', tm value is 59 ℃;
the primer pair can specifically amplify fragments containing the rs58928048 insertion deletion site polymorphism in METTL genes.
The total volume of the PCR reaction system was 10uL, which includes: 1. Mu.L of DNA template, 50. Mu.M of specific primer pair each 0.04. Mu.L, 2.5U/. Mu.L Taq DNA polymerase 0.08. Mu.L; 0.2. Mu.L of 2.5mM dNTP mix; 0.6. Mu.L of 25mM MgCl 2 solution; 1. Mu.L of 10 XPCR reaction buffer; the deionized water is replenished;
The reaction was performed on Eppendorf Mastercycler nexus PCR amplification apparatus under the following conditions: 94 ℃ for 3min; a further 30 PCR cycles were performed: 94 ℃ for 30s,59 ℃ for 30s, and 72 ℃ for 1min; finally, the temperature is 72 ℃ for 5min.
Step 3: indel genotyping
And (3) performing capillary electrophoresis separation on the amplified product by using an ABI 3500 gene sequencer to obtain the genotype of the detected individual, and providing explanation by a professional.
Experimental results of the invention:
the differences in gene frequency and risk of disease OR values at this site for the control and SCD case groups are shown in table 1.
TABLE 1 correlation between polymorphic site rs58928048 and SCD risk of developing disease
Note that: CI confidence interval; OR ratio; a Correction according to age and sex
As shown in table 1, within the 95% confidence interval, genotype of the deletion/deletion type individual was 0.45 times the risk of developing SCD than the insertion/insertion type individual, and genotype of the insertion/deletion type individual was 0.69 times the risk of developing SCD than the insertion/insertion type individual in the co-dominant model; in the recessive model, individuals with genotype that is deleted/absent are at 0.56 times the risk of developing SCD than individuals with insert/insert or insert/absent; in the additive model, individuals carrying the deletion allele are at 0.69 times the risk of developing SCD than individuals carrying the insertion allele.
The gene sequencing diagram and SDS-PAGE gel electrophoresis typing diagram are shown in FIG. 1. Wherein FIG. 1A is an example of a template strand sequencing result, corresponding to a six base insertion deletion of the coding strand at rs58928048 at the underline; FIG. 1B shows the result of electrophoresis of 14 DNA samples from different individuals using the PCR amplification system of the present invention, 2, 3, 11, 12 being insert homozygotes, 1, 4, 7, 13 being deletion homozygotes, the remainder being heterozygotes; "×", DNA MARKER.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Sequence listing
<110> University of Suzhou
<120> Molecular marker and kit for predicting sudden cardiac death based on METTL gene polymorphism site
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 1
cacatgaatg cttggactag aat 23
<210> 2
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 2
gtgtattttt tttcctggag aga 23
Claims (3)
1. The application of a primer for amplifying METTL gene rs58928048 locus in preparing a sudden cardiac death susceptibility detection kit is characterized in that: the sudden cardiac death susceptibility detection kit is used for detecting the genotype of METTL gene rs58928048 locus.
2. The use according to claim 1, characterized in that: the primer for amplifying METTL gene rs58928048 locus comprises a sense primer and an antisense primer, wherein the nucleotide sequence of the sense primer is shown as SEQ ID NO.1, and the nucleotide sequence of the antisense primer is shown as SEQ ID NO. 2.
3. The use according to claim 1, characterized in that: the sudden cardiac death susceptibility detection kit also comprises components for PCR amplification and capillary electrophoresis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210521337.1A CN114736960B (en) | 2022-05-13 | 2022-05-13 | Molecular marker and kit for predicting sudden cardiac death based on METTL gene polymorphism sites |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210521337.1A CN114736960B (en) | 2022-05-13 | 2022-05-13 | Molecular marker and kit for predicting sudden cardiac death based on METTL gene polymorphism sites |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114736960A CN114736960A (en) | 2022-07-12 |
| CN114736960B true CN114736960B (en) | 2024-05-31 |
Family
ID=82285563
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210521337.1A Active CN114736960B (en) | 2022-05-13 | 2022-05-13 | Molecular marker and kit for predicting sudden cardiac death based on METTL gene polymorphism sites |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114736960B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115433776B (en) * | 2022-09-30 | 2023-12-22 | 中国医学科学院阜外医院 | Application of CCN3 in regulating vascular smooth muscle cell calcification |
| CN116716391A (en) * | 2023-04-26 | 2023-09-08 | 苏州大学 | Molecular marker and kit for detecting sudden cardiac death by using TBX5 gene polymorphism sites |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112111572A (en) * | 2020-10-12 | 2020-12-22 | 苏州大学 | STAT5A gene insertion deletion polymorphism site-based sudden cardiac death susceptibility detection kit |
-
2022
- 2022-05-13 CN CN202210521337.1A patent/CN114736960B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112111572A (en) * | 2020-10-12 | 2020-12-22 | 苏州大学 | STAT5A gene insertion deletion polymorphism site-based sudden cardiac death susceptibility detection kit |
| WO2022077539A1 (en) * | 2020-10-12 | 2022-04-21 | 苏州大学 | Susceptibility detection kit for sudden cardiac death based on insertion-deletion polymorphic site of stat5a gene |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114736960A (en) | 2022-07-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114736960B (en) | Molecular marker and kit for predicting sudden cardiac death based on METTL gene polymorphism sites | |
| Jakupciak et al. | Mitochondrial DNA as a cancer biomarker | |
| CN107858423B (en) | Kit for predicting susceptibility to sudden cardiac death | |
| WO2014136870A1 (en) | Simple detection method for rna modification, and method for detecting type-ii diabetes using said detection method | |
| Jasinska et al. | A non-human primate system for large-scale genetic studies of complex traits | |
| WO2009104730A1 (en) | Identification of hypertension susceptibility gene group | |
| CN112111572B (en) | STAT5A gene insertion deletion polymorphism site-based sudden cardiac death susceptibility detection kit | |
| Pichon et al. | Analysis and annotation of DNA methylation in two nonhuman primate species using the Infinium Human Methylation 450K and EPIC BeadChips | |
| Sponholz et al. | Polymorphisms of cystathionine beta-synthase gene are associated with susceptibility to sepsis | |
| JP2011528554A5 (en) | ||
| CN112029853B (en) | Sudden cardiac death susceptibility detection kit based on HSPA1B gene insertion deletion polymorphic site | |
| JP2009118803A (en) | Method for determination of the risk of developing obesity based on gene polymorphism associated with human body fat mass | |
| JP4491276B2 (en) | Method and kit for detecting the presence of a single nucleotide polymorphism in a target DNA sequence | |
| CN112159841B (en) | Sudden cardiac death susceptibility detection kit based on COX10 gene insertion deletion polymorphic site | |
| CN116426632A (en) | Molecular marker and kit for detecting sudden cardiac death by LTBP4 gene polymorphism site | |
| Jasinska | Biological Resources for Genomic Investigation in the Vervet Monkey (Chlorocebus) | |
| EP3221471B1 (en) | Method for predicting increased resistance of a rainbow trout to infectious pancreatic necrosis (ipn) | |
| Mannen et al. | Development and mapping of microsatellite markers derived from cDNA in Japanese quail (Coturnix japonica) | |
| CN115725745B (en) | SNP molecular marker related to sheep multi-thoracic vertebrae and amplification primer set and application | |
| WO2022077540A1 (en) | Sudden cardiac death susceptibility detection kit based on stim1 gene insertion and deletion polymorphic sites | |
| KR102565803B1 (en) | Method for providing information for hypertension and kits using the same | |
| JP4505839B2 (en) | CYP2D6 * 4 mutation detection method and nucleic acid probe and kit therefor | |
| CN116716391A (en) | Molecular marker and kit for detecting sudden cardiac death by using TBX5 gene polymorphism sites | |
| CN114622020B (en) | KLHL31 gene molecular marker related to chicken growth traits and application thereof | |
| JP4998874B2 (en) | Determination method of inflammatory diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |