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Method and kit for detecting polymorphism of mitochondrial ND1 gene mononucleotide, and application of kit

A single nucleotide polymorphism and mitochondrial technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problem of increasing PCR product contamination, cannot achieve closed-tube operation, and is difficult to separate Type and other problems, to achieve the effect of low detection cost, convenient and simple detection, and easy operation

Inactive Publication Date: 2010-07-14
卫生部北京医院
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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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  • Method and kit for detecting polymorphism of mitochondrial ND1 gene mononucleotide, and application of kit
  • Method and kit for detecting polymorphism of mitochondrial ND1 gene mononucleotide, and application of kit
  • Method and kit for detecting polymorphism of mitochondrial ND1 gene mononucleotide, and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Blood Sample Collection and Genomic DNA Extraction

[0062] 1. Sample selection

[0063] 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 358 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.

[0064] 2. Preparation of Genomic DNA

[0065] 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 5ml of cell lysate (containing 10mmol / L Tris-HCl pH8.0, 10mmol / L EDTA pH8.0, 15mmol / L Nacl, 0.4% SDS, 0.1mg / ml proteinase K) and incubate overnight at 37°C. Then u...

Embodiment 2

[0066] Embodiment 2: Identification and determination of SNP

[0067] The invention adopts HRMA and PCR sequencing technology to simultaneously detect the C3970T site (the allelic site pair is A / G).

[0068] 1. Specific primers are as follows:

[0069] F1: 5'-CTAGTCTCAGGCTTCAACATC-3' (SEQ ID NO.1)

[0070] R1: 5'-TGTTTGTGTATTCGGCTAT-3' (SEQ ID NO.2)

[0071] 2. Amplify some fragments near C3970T by PCR; prepare mixed solution: add 2 ul of genomic DNA solution, 2 ul of 10X PCR buffer, 0.4 ul of 10mM dNTP, 2 ul of Taq DNA polymerase, respectively 0.2 ul of the above step 1) in Example 1 For each primer described, 2ul LCGreen PLUS+ saturated fluorescent dye, 0.1ul C1 and C2 oligonucleotide internal references respectively. 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, 56°C for 30 seconds, 72°C for 3 s, and 2°C for 7 minutes for 35 cycles. After the reaction was completed, the PCR product was sub...

Embodiment 3

[0075] Example 3: Association of mtDNA 3970 single nucleotide polymorphisms with aging-related degenerative diseases

[0076] 1. Statistical method: use STATA8.0 and SPSS11.0 software Yates chi-square test to calculate the carrier frequency of mtDNA 3970 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).

[0077] 2. Results

[0078] 1. Distribution of mtDNA 3970 single nucleotide polymorphisms in healthy people

[0079] According to the method of Examples 1 and 2, the gene polymorphisms of 362 healthy people (without cardiovascular and cerebrovascular diseases, cancer, etc., age≤60 years old) were determined. 285 people had C polymorphism at base 3970 (78.1%), and 77 people had T polymorphism (21.3%).

[0080] 2. Distribution of mt...

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Abstract

The invention discloses a method for detecting SNP of mitochondrial ND1 gene mt DNA 3970 locus, a kit for detecting the SNP of the locus and application of the kit. The method does not have special restriction on samples, namely, either body fluid or histiocyte can be used as a detection sample, so the detection is convenient and rapid; auele specific primers designed by the method do not have strict requirement on the length of basic group of a region to be amplified, and PCR products can be obtained and high-resolution melting curve can be analyzed by using the auele specific primers; and before PCR reaction, saturated fluorescent dye is added, a light scanner detects fluorescent signals by optics and draws a temperature melting curve, so that wild type, heterozygous mutation and homozygous mutation can be accurately differentiated according to the curve. The invention has the advantages of simple and convenient operation, low detection cost, accurate detection result, and high application and market values.

Description

technical field [0001] The invention relates to a method for detecting the single nucleotide polymorphism (SNP) of the mitochondrial ND1 gene, in particular to a method for detecting the SNP at the mtDNA 3970 site of the mitochondrial ND1 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...

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相蕾史晓红唐雷孙亮
Owner 卫生部北京医院
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