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Detection kit and method for polymorphism of ALDH2 gene RS671

A detection kit and detection method technology, applied in the field of genetic detection, can solve the problems of high cost and expensive sequencing equipment, and achieve the effects of strong compliance, high sensitivity and convenient use

Inactive Publication Date: 2018-12-21
GUANGXI IGE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The need to meet the above three points at the same time has brought great challenges to the design of the experiment; patent CN201710153613, using the second generation sequencing technology to detect the RS671 site, NGS technology is a high-throughput technology developed in recent years Detection technology, multiple samples can be mixed and sequenced to reduce costs, but for the detection of a single sample, the entire detection process includes 1) genomic DNA extraction 2) library construction and adapter 3) library quality control and sequencing 4) analysis of disembarkation data, and or Ion (PGM) sequencing instruments are very expensive, the cost is extremely high, this method is not suitable for SNP detection of a single gene

Method used

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  • Detection kit and method for polymorphism of ALDH2 gene RS671
  • Detection kit and method for polymorphism of ALDH2 gene RS671
  • Detection kit and method for polymorphism of ALDH2 gene RS671

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Detection of Aldehyde Dehydrogenase ALDH2 Gene RS671 Polymorphism

[0038] The ALDH2 gene RS671 polymorphism detection kit described in this embodiment includes primers and probes for the detection of acetaldehyde dehydrogenase ALDH2 gene mutation, as shown in Table 1:

[0039]Table 1 Primers and probes for detection of aldehyde dehydrogenase ALDH2 gene mutation

[0040]

[0041] The kit also includes control DNA; the control DNA includes ALDH2 gene RS671 site AA homozygous, GG homozygous and GA heterozygous, the nucleotide sequence (SEQ ID NO.10) of the ALDH2 gene RS671 site is as follows:

[0042]

[0043] Among them, AA homozygous R is A (A:T); GG homozygous R is G (G:C); GA heterozygous R: is G / A (allele G:C / A:T).

[0044] Sample acquisition:

[0045] Use a saliva collector, put it near your mouth to collect 2ml of saliva, add cell preservation solution, cover the lid, and shake the collection tube up and down or left and right for 30 seconds, so t...

Embodiment 2

[0057] Example 2 Different primer pairs or probes are used to detect differences in RS671

[0058] Using the RS671 site of the acetaldehyde dehydrogenase ALDH2 gene, design a specific primer sequence, use the forward or reverse complementary sequence of the target sequence where the mutation site is located as a template, and design a probe sequence for the site, including the present invention For the sequences of SEQ ID NO.1 and SEQ ID NO.2 in Example 2, all designed alternative primer pairs are shown in Table 2, and the probes are shown in Table 3.

[0059] Table 3 Primer Sequence

[0060]

[0061] Table 4 Probe sequence

[0062]

[0063] According to the steps described in Example 2, a DNA sample was obtained, a reaction solution was prepared, and acetaldehyde dehydrogenase ALDH2 gene RS671 was detected.

[0064] Wherein, the primer combination is: RS671-1's SEQ ID NO.1 and SEQ ID NO.2 are a group, RS671-2's SEQ ID NO.3 and SEQ ID NO.4 are a group, RS671-3's SEQ ID...

Embodiment 4

[0066] Example 4 Concentration Comparison of Upstream and Downstream Primers in PCR Amplification to Detect Differences in RS671

[0067] After the DNA samples were obtained according to the steps described in Example 2, PCR amplification reaction systems with different concentrations of upstream and downstream primers were prepared respectively.

[0068] PCR amplification reaction system one (upstream primer: downstream primer = 1:1):

[0069] 1×master mix 23μl, DNA 2μl, HS taq DNA polymerase 1U; among them, 1×master mix is ​​prepared from the following components:

[0070]

[0071] PCR amplification reaction system two (upstream primer: downstream primer = 2:1):

[0072] 1×master mix 23μl, DNA 2μl, HS taq DNA polymerase 1U; among them, 1×master mix is ​​prepared from the following components:

[0073]

[0074]

[0075] PCR amplification reaction system three (upstream primer: downstream primer = 10:1):

[0076] 1×master mix 23μl, DNA 2μl, HS taq DNA polymerase 1U...

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Abstract

The invention provides a detection kit for polymorphism of an ALDH2 gene RS671. The detection kit comprises primers and a probe for the ALDH2 gene RS671; nucleotide sequences of the primers are shownin SEQ ID NO.1 and SEQ ID NO.2; the nucleotide sequence of the probe is shown in SEQ ID NO.7, a fluorophore is labeled at the 5' end of the probe, and a quenching group is labeled at the 3' end; the reasonable proportion of upstream and downstream primers is controlled, the polymorphism of the ALDH2 gene RS671 in to-be-detected sample DNA is detected and analyzed in combination with PCR amplification and melting curve methods, and the detection kit is high in resolution of G / A difference analysis of the RS671 site, reasonable in cost and simple and convenient to operate.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to a detection kit and method for RS671 polymorphism of ALDH2 gene. Background technique [0002] Acetaldehyde dehydrogenase (ALDH for short) (EC1.2.1.10) (CAS[9028-91-5]), a type of aldehyde dehydrogenase, is responsible for catalyzing the oxidation of acetaldehyde to acetic acid. [0003] Alcohol dehydrogenase in the liver is responsible for oxidizing ethanol (a component of wine) into acetaldehyde, and the resulting acetaldehyde is used as a substrate to be further converted into harmless acetic acid (ie a component of vinegar) under the catalysis of acetaldehyde dehydrogenase. Acetaldehyde is more toxic than ethanol and is one of the main causes of hangovers. Moreover, acetaldehyde is suspected to be carcinogenic, and it has a certain relationship with the occurrence of human tumors. The main responsible for the conversion of acetaldehyde in the human body is the acetaldehyde deh...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858
CPCC12Q1/6858C12Q1/6886C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 姬宏超
Owner GUANGXI IGE BIOTECH
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