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CYP2A6 gene polymorphism detection specific primers and liquid chip

A gene polymorphism and detection solution technology, applied in the field of molecular biology, can solve the problems of unsatisfactory practical application and unusability, and achieve the effects of avoiding uncertain factors, consistent detection results, and low cross-reaction rate

Inactive Publication Date: 2013-02-06
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The PCR-RFLP method is based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of a new site, a specific fragment is amplified by PCR, and then cut and amplified with a restriction endonuclease. Amplify the product and observe the size of the fragment by electrophoresis. This method is used to detect gene mutations with altered restriction sites, and can directly determine the genotype. However, this method cannot be used for the detection of gene mutations without new restriction sites.
Again, the above methods all have limitations in detection throughput, and can only detect one mutation type at a time, which cannot meet the needs of practical applications.

Method used

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  • CYP2A6 gene polymorphism detection specific primers and liquid chip
  • CYP2A6 gene polymorphism detection specific primers and liquid chip
  • CYP2A6 gene polymorphism detection specific primers and liquid chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 CYP2A6 gene polymorphism detection liquid chip mainly includes:

[0024] 1. ASPE Primers

[0025] Specific primer sequences were designed for wild-type and mutant types of eight common genotypes of CYP2A6 gene, G84T, G133A, T229A, T106C, G148T, T149G, T53C and G108A. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0026] ASPE primer sequence (Tag sequence+specific primer sequence) of table 1 CYP2A6 gene

[0027]

[0028]

[0029]Each ASPE primer includes two parts, the 5' end is a specific tag sequence for the anti-tag sequence on the corresponding microsphere, and the 3' end is a mutant or wild-type specific primer fragment (as shown in the above table 1). All ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized primer was prepared into a stock solution of 100 pmol / mL with 10 mmol / L Tris Buffer.

[0030] 2. Microspheres ...

Embodiment 2

[0041] Example 2 Detection of samples using the CYP2A6 gene polymorphism detection liquid chip described in Example 1

[0042] The formula of described various solutions is as follows:

[0043] 50mM MES buffer (pH5.0) formula (250ml):

[0044]

[0045] 2×Tm hybridization buffer

[0046] Reagent

source

Final concentration

Dosage per 250ml

1M Tris-HCl, pH8.0

SigmaT3038

0.2M

50ml

5MNaCl

Sigma S5150

0.4M

20ml

Triton X-100

Sigma T8787

0.16％

0.4ml

[0047] Store at 4°C after filtration.

[0048] ExoSAP-IT kit was purchased from US USB Company.

[0049] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0050] 1. Sample DNA extraction:

[0051] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0052] 2. PCR amplification of samples to be tested

[0053] Six pairs of primer...

Embodiment 3

[0098] Example 3 Detection of CYP2A6 gene polymorphism site by liquid chip with different ASPE primers

[0099] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0100] Taking the CYP2A6 gene T106C and T149G site mutation detection liquid chip as an example, the specific primer sequence of the 3' end of the ASPE primer was designed for the wild type and mutant type of T106C and T149G, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.16, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.33-SEQ ID NO.48. The specific design is shown in the following table (Table 8). The synthesis of ASPE primers, microspheres coated with anti-tag sequences, amplification primers, detection methods, etc. are as described in Example 1 and Example 2.

[0101] Table 8 Design of liquid phase chip preparation

[0102]...

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Abstract

The invention discloses CYP2A6 gene detection specific primers and a liquid chip. The liquid chip mainly comprises: various ASPE primers composed of 5' terminal tag sequence and 3' terminal specific primer sequences aiming at target gene mutation site, microspheres coated with anti-tag sequence, and amplification primers. The specific primer sequences comprise: SEQ ID NO.17 and SEQ ID NO.18 aiming at G84T site; SEQ IDNO.19 and SEQ ID NO.20 aiming at G133A site; SEQ ID NO.21 and SEQ ID NO.22 aiming at T229A site; SEQ ID NO.23 and SEQ ID NO.24 aiming at T106C site; SEQ ID NO.25 and SEQ ID NO.26 aiming at G148T site; SEQ ID NO.27 and SEQ ID NO.28 aiming at T149G site; SEQ ID NO.29 and SEQ IDNO.30 aiming at T53C site; and / or SEQ ID NO.31 and SEQ ID NO.32 aiming at G108A site. The matching rate of the detection result obtained by using the liquid chip provided by the invention and a result obtained by using a sequencing method is 100%. Therefore, wild-type and mutant-type parallel detections of a plurality of mutation sites are realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a CYP2A6 gene polymorphism detection specific primer and a liquid phase chip. Background technique [0002] In recent years, research on the relationship between metabolic enzyme gene polymorphisms and tumor susceptibility has become a hot topic in the field of tumor research. The CYP II family is the family with the largest number of known CYP isozymes and the most complex structure. Among them, the CYP2A gene family is located between 19q12 and 19q13.2 on the long arm of human chromosome 19, with a size of about 350kb. It includes 3 complete genes: CYP2A6, CYP2A13, and CYP2A7, and two CYP2A7 pseudogenes. These genes all have a typical CYP2A gene subfamily structure with 9 exons. Among them, CYP2A6 is the most important member, encoding coumarin 7-hydroxylase (eoumarin7-hydroxide), which plays an important role in the metabolism of many e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 许嘉森杨惠夷
Owner SUREXAM BIO TECH
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