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SNP G10320A molecular marker of mitochondrion ND3 gene of senescence-associated degenerative disease, detecting method and kit thereof

A technique for degenerative diseases and molecular markers, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of increased PCR product contamination, inability to achieve closed-tube operation, cumbersome work, etc.

Inactive Publication Date: 2009-09-09
BEIJING HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the latter two methods are difficult to type for some wild-type and homozygous mutations
Although a known homozygous sample can be mixed with an unknown sample to form a heterologous chain for typing, this will repeat the experimental process twice, the first time to distinguish heterozygous and homozygous variants, and the second time Known homozygous samples are mixed with unknown samples, making the job more tedious
At the same time, the closed-tube operation cannot be achieved in the two operations, which increases the contamination of PCR products.

Method used

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  • SNP G10320A molecular marker of mitochondrion ND3 gene of senescence-associated degenerative disease, detecting method and kit thereof
  • SNP G10320A molecular marker of mitochondrion ND3 gene of senescence-associated degenerative disease, detecting method and kit thereof
  • SNP G10320A molecular marker of mitochondrion ND3 gene of senescence-associated degenerative disease, detecting method and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Blood Sample Collection and Genomic DNA Extraction

[0067] 1. Sample selection

[0068] Household survey of long-lived elderly in Bama, Guangxi, aged ≥ 90 years old. Clear consciousness, independent life, able to cooperate with examination, no cardiovascular and cerebrovascular diseases, Alzheimer's disease, Parkinson's disease, cancer and other aging-related degenerative diseases, a total of 372 cases. The basic information of all subjects was recorded and informed consent was signed by themselves or their relatives. This study was also approved by the institutional ethics committee.

[0069] 2. Preparation of Genomic DNA

[0070] In the presence of the anticoagulant EDTA, 5 ml of peripheral blood collected from the subject was centrifuged at 2500 rpm for 30 minutes to remove serum. Then add 5 ml of cell lysate (containing 10 mmol / L Tris-HCl pH8.0, 10 mmol / L EDTA pH8.0, 15 mmol / L Nacl, 0.4% SDS, 0.1 mg / ml proteinase K) and incubate overnight at 37°C. T...

Embodiment 2

[0071] Embodiment 2: Identification and determination of SNP

[0072] The invention adopts HRMA and PCR sequencing technology to simultaneously detect the G10320A site (the allelic site pair is C / T).

[0073] 1. Specific primers are as follows:

[0074] F1: 5'-ATTTGATCTAGAAATTGCCCTCC-3'; (SEQ ID NO.2)

[0075] R1: 5'-TTGTAGTCACTCATAGGCCAG-3' (SEQ ID NO.3)

[0076] 2. Amplify some fragments near G10320A by PCR; prepare mixed solution: add 2ul of genomic DNA solution, 2ul of 10X PCR buffer, 0.4ul of 10mM dNTP, 2ul of Taq DNA polymerase, and 0.2ul of F1 and R1 respectively in Example 1 For sense primers and antisense primers, 2ul LCGreen PLUS+ saturated fluorescent dye, 0.2ul C1 probe. Next, pure water was added to make the total volume 20ul. The reaction was performed at 95°C for 5 minutes, 95°C for 1 minute, 63.5°C for 30 seconds, 72°C for 10 seconds, and 72°C for 7 minutes for 35 cycles. After the reaction was completed, the PCR product was subjected to two further cycles...

Embodiment 3

[0080] Example 3: Association of mtDNA 10320 single nucleotide polymorphisms with aging-related degenerative diseases

[0081] 1. Statistical method: use the Pearson chi-square test in STATA8.0 and SPSS11.0 software to calculate the carrier frequency of mtDNA 10320 single nucleotide polymorphism, carry out continuous correction and one-sided asymptotic probability analysis, statistical significance Levels were set at P<0.05. Single factor Logistic regression analysis was used to calculate the risk OR value of longevity and its 95% confidence interval (CI).

[0082] 2. Results

[0083] 1. Distribution of mtDNA 10320 single nucleotide polymorphisms in healthy people

[0084] According to the methods of Examples 1 and 2, the gene polymorphisms of 384 healthy people (age≤60 years old) were determined. 379 people had G polymorphism at base 10320 (98.7%), and 5 people had A polymorphism (1.3%).

[0085] 2. Distribution of mtDNA 10320 single nucleotide polymorphisms in healthy el...

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Abstract

The present invention discloses a molecular marker of senescence-associated degenerative disease, a method for detecting the molecular marker SNP and a detecting kit thereof. The method of the invention has no special restriction to the sample. The body fluid and histiocyte can be equally taken as the detecting sample of the invention to cause that the detection is convenient and concise. The specific primer designed by the invention has no strict requirement for the length of basic group in the amplification area. The PCR product of the invention can be obtained through the specific primer for analyzing the high resolution fluxing curve. The saturated fluorescent dye is added before PCR reaction. A light-scanner detects the variation of fluorescent signal through optical detection and draws a temperature fluxing curve. The wild type, heterozygous mutation and homozygous mutation can be accurately differentiated according to the curve. The invention has the advantages of easy operation, low detection cost, accurate detection result, excellent application value and market value.

Description

technical field [0001] The invention relates to a method for detecting the single nucleotide polymorphism (SNP) of the mitochondrial ND3 gene, specifically a method for detecting the SNP at the mtDNA 10320 site of the mitochondrial ND3 gene and a kit for detecting the SNP at the site and Application of the kit. Background technique [0002] Health and longevity has always been a research hotspot in various countries. This is because the long-lived elderly do not suffer from aging-related degenerative diseases such as stroke, cardiovascular disease, type 2 diabetes, Parkinson's disease, senile dementia, cancer, etc. during the aging process. And healthy life. These aging-related degenerative diseases not only bring a heavy economic burden to patients and affect the quality of life, but also since my country entered an aging society in 1998, the proportion of the elderly population in the total population (10%) has been increasing year by year. In 2020, the population over th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 杨泽冯洁孔放张建佚陈静万钢齐科研唐雷
Owner BEIJING HOSPITAL
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