Primer pair and kit for detecting ALDH2 (Aldehyde Dehydrogenase 2) genotype with pyrosequencing method

A technology of pyrosequencing and genotyping, which is applied in the field of primer pairs and kits for detecting genotyping of acetaldehyde dehydrogenase 2 by pyrosequencing, and can solve the problem of difficulty in meeting clinical testing requirements and inaccurate testing results. High, long detection cycle and other issues, to achieve the effects of fast sequencing, high-throughput sample detection, and short reaction time

Inactive Publication Date: 2015-12-23
CHANGSHA 3G BIOTECH
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

[0020] In order to solve the technical problems of low accuracy of detection results, long detection period, cumbersome operation and difficulty in meeting the requirements of clinical examination in the process of detecting ALDH2 genotyping by the above method, the present invention provides an Primer pair and kit for detection of ALDH2 genotyping by pyrosequencing method that is short, easy to operate and effectively meets the requirements of clinical testing

Method used

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  • Primer pair and kit for detecting ALDH2 (Aldehyde Dehydrogenase 2) genotype with pyrosequencing method
  • Primer pair and kit for detecting ALDH2 (Aldehyde Dehydrogenase 2) genotype with pyrosequencing method
  • Primer pair and kit for detecting ALDH2 (Aldehyde Dehydrogenase 2) genotype with pyrosequencing method

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Experimental program
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Effect test

Embodiment 1

[0052] Embodiment 1: the preparation of kit

[0053] 1. Design and synthesis of primers and probes

[0054] For the G1510A polymorphic site where the human ALDH2 gene mutation is located in the 12th exon, select the specific mutation site, and use the PyroMarkAssayDesign2.0 software to design primers; the forward amplification primer, reverse amplification primer and sequencing primer Purified by PAGE first, and then purified by HPLC, wherein the 5' of SEQ ID NO.1 is labeled with biotin.

[0055] Table 1. Mutation site and type:

[0056] Mutation

base change

ALDH2*2

ATACACTG / AAAGTGAAA

[0057] The amplified sequence is shown in Table 2:

[0058] Table 2. Specific amplification primers and primer sequences

[0059]

[0060] 2. Selection of reference substance

[0061] A synthetic oligonucleotide chain TAYGGTTTGCAcontrololigo was used as the quality control substance; DNase / RNase-Free water was used as the blank control substance.

[0062]...

Embodiment 2

[0065] Embodiment 2: the use of kit

[0066] 1. Sample testing

[0067] Dissolve the dry powder of the primers (the validity period of the primers is 1 month after dissolution). Prepare the system according to the number of templates: take the PCR reaction solution, add dissolved primers, uracil DNA glycosylase, and TaqDNA polymerase, aliquot the system, add sample DNA, blank control substance or positive control substance as templates, and form a PCR reaction system. Perform PCR amplification according to the PCR reaction procedure.

[0068] The main components of the ALDH2*2 system are as follows:

[0069] Table 4. Main components of ALDH2*2 system

[0070]

[0071] The system reaction procedure is as follows:

[0072] Table 5. PCR reaction program

[0073]

[0074] After the amplification is completed, check the PCR results on agarose gel to proceed to the next step.

[0075] 2. Pyrosequencing

[0076] Sequencing was performed in accordance with the standard o...

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Abstract

The invention relates to a sequencing primer pair for detecting an ALDH2 (Aldehyde Dehydrogenase 2) genotype with a pyrosequencing method and a kit thereof, and belongs to the technical field of in-vitro nucleic acid detection. The primer pair comprises a positive amplification primer, a reverse amplification primer and a sequencing primer, wherein a 5' end of the positive amplifier primer is subjected to biotin labeling. The kit comprises the positive amplification primer, PCR (Polymerase Chain Reaction) reaction liquid containing the reverse amplification primer, the sequencing primer, uracil DNA (Deoxyribose Nucleic Acid) glycosylase and Taq polymerase. The kit provided by the invention has the advantages of accurate detection result, high specificity, short detection period, easiness in operation, and capability of effectively meeting clinical examination requirements. Moreover, the kit further has the advantages that a reaction process can be monitored in real time, the reaction time is short, a PCR product can be subjected to pyrosequencing by a pyrosequencing instrument and high-flux sample detection through simple treatment, and the sensitivity is higher compared with a golden standard method, namely, a capillary electrophoresis sequencing method.

Description

technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a primer pair and a kit for detecting acetaldehyde dehydrogenase 2 (ALDH2) genotyping by pyrosequencing. Background technique [0002] Human acetaldehyde dehydrogenase (Aldehydehydrogenasegene, ALDH) is a tetrameric protein that catalyzes the oxidation of acetaldehyde and other aliphatic aldehydes. At present, it has been found that there are 19 isoenzymes of ALDH, mainly including ALDH1-4, among which ALDH2 is the most important. It is highly expressed in the liver and stomach, and is one of the most important enzymes in the pathway of ethanol metabolism. [0003] The ALDH2 gene is located on human chromosome 12 (12q24.2), and its main polymorphism is rs671, which is G1510A located in exon 12. Due to the G1510A polymorphism in the ALDH2 gene, the glutamic acid at position 487 of the amino acid sequence is replaced by lysine (ie, Glu487Lys), and the wild...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6869
Inventor 滕祥云周姗
Owner CHANGSHA 3G BIOTECH
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