Specific primer and liquid chip for detecting polymorphism of NAT1 (N-acetyltransferase 1) gene

A technology of gene polymorphism and detection liquid, applied in the field of molecular biology, can solve the problems that cannot meet the practical application and cannot be used, and achieve the effect of avoiding uncertain factors, consistent detection effect and low cross-reaction rate.

Inactive Publication Date: 2013-03-06
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 ...

Method used

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  • Specific primer and liquid chip for detecting polymorphism of NAT1 (N-acetyltransferase 1) gene
  • Specific primer and liquid chip for detecting polymorphism of NAT1 (N-acetyltransferase 1) gene
  • Specific primer and liquid chip for detecting polymorphism of NAT1 (N-acetyltransferase 1) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The NAT1 gene polymorphism detection liquid chip described in this embodiment mainly includes:

[0028] 1. ASPE Primers

[0029] Specific primer sequences were designed for wild-type and mutant types of seven common genotypes of NAT1 gene, C135T, G136A, T216G, A328T, A460T, C106T and C199T. ASPE primers consist of "Tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0030] Table 1 ASPE primer sequence (Tag sequence + specific primer sequence) of NAT1 gene

[0031]

[0032]

[0033] 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.

[003...

Embodiment 3

[0102] The liquid phase chip of embodiment 3 different ASPE primers detects the polymorphic site of NAT1 gene

[0103] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0104] Taking the NAT1 gene G136A and A328T 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 G136A and A328T, and the Tag sequence at the 5' end of the ASPE primer was selected from SEQ ID NO.1-SEQ ID NO.14, correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.29-SEQ ID NO.42. 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.

[0105] Table 8 Design of liquid phase chip preparation

[...

Embodiment 4

[0116] Example 4 Selection of Specific Primer Sequences for NAT1 Gene Polymorphism Detection

[0117] 1. Design of liquid-phase chip preparation (selection of wild-type and mutant-specific primer sequences)

[0118] Taking the polymorphic site detection liquid chip of NAT1 gene T216G and A460T as an example, using the forward or reverse complementary sequence of the target sequence where the mutation site is located as a template, the wild type and mutant type of T216G and A460T were designed respectively The specific primer sequences at the 3' end of the ASPE primers include the preferred specific primer sequences and 2 alternative specific primer sequences in Example 1 of the present invention, as shown in Table 11. in, Inner bases are polymorphic sites.

[0119] Table 11 specific primer sequence

[0120]

[0121]

[0122] Taking the polymorphic site detection liquid chip of NAT1 gene T216G and A460T as an example, different specific primer sequences were selected ...

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Abstract

The invention discloses a liquid chip and specific primer for detecting an NAT1 (N-acetyltransferase 1) gene. The liquid chip mainly comprises ASPE (allele specific primer extension) primers, microspheres and amplification primers, wherein each ASPE primer is formed by tag sequences at the 5' terminal and specific primer sequences which are arranged at the 3' terminal and aim at the mutation site of the target gene; the specific primer sequences are SEQ ID NO.15 and SEQ ID NO.16 aiming at the C135T site, SEQ ID NO.17 and SEQ ID NO.18 aiming at the G136A site, SEQ ID NO.19 and SEQ ID NO.20 aiming at the T216G site, SEQ ID NO.21 and SEQ ID NO.22 aiming at the A328T site, SEQ ID NO.23 and SEQ ID NO.24 aiming at the A460T site, SEQ ID NO.25 and SEQ ID NO.26 aiming at the C106T site and/or SEQ ID NO.27 and SEQ ID NO.28 aiming at the C199T site; and the microspheres are enveloped by anti-tag sequences. The detecting liquid chip provided by the invention has the advantages that the consistency of the detection result and the sequencing method is as high as 100%, thus parallel detection of wild types and mutant types of a plurality of mutation sites is achieved.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a specific primer for NAT1 gene polymorphism detection and a liquid phase chip. Background technique [0002] N-acetylation metabolism is an important biotransformation pathway for many arylamines and hydrazines, catalyzed by N-acetyltransferase (NAT) dependent on acetyl-CoA, N-acetyltransferase (N-acetyltrans ferasel , NAT) is an enzyme system that catalyzes the acetylation of nitrogen-containing compounds in the body. It can activate or degrade nitrogen-containing exogenous substances, especially exogenous carcinogens containing amine groups. The genes encoding NAT are NAT1 and NAT2, which are located on the 8th pair of chromosomes (8p21.3-23.1). Studies have shown that human NAT1 has a wider tissue distribution and exists in colonic mucosa, liver, bladder, red blood cells and lymphocytes , is the dominant acetyltransferase in colorectal ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11G01N21/64
Inventor 许嘉森吴诗扬
Owner SUREXAM BIO TECH
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