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TGM5 gene mutation detection specific primer and liquid phase chip

A technology of TGM5 and detection solution, applied in the field of molecular biology, can solve the problems of low degree of automation and many manual operations, and achieve the effect of simple steps, avoiding cross-reaction, and consistent detection effect.

Active Publication Date: 2013-12-18
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the TGM5 gene mutation detection methods mainly include: Illumina fiber optic microbead chip technology, although Illumina fiber optic microbead chip technology is a high-throughput detection system with high sensitivity and accuracy, but the degree of automation is low, and there are many manual operations, which is difficult to meet the actual conditions. application needs

Method used

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  • TGM5 gene mutation detection specific primer and liquid phase chip
  • TGM5 gene mutation detection specific primer and liquid phase chip
  • TGM5 gene mutation detection specific primer and liquid phase chip

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Embodiment 1

[0044] Embodiment 1 TGM5 gene mutation detection liquid chip mainly includes:

[0045] 1. ASPE Primers

[0046] Specific primer sequences were designed for wild-type and mutant types of TGM5 gene three common genotypes T116C, A212G and A117G. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0047] The ASPE primer sequence (tag sequence+specific primer sequence) of table 1TGM5 gene

[0048]

[0049] 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.

[0050] 2. Microspheres coated with anti-tag sequences

[0051] According to ...

Embodiment 2

[0063] Example 2 Detection of samples using the TGM5 gene mutation detection liquid chip described in Example 1

[0064] The formula of described various solutions is as follows:

[0065] 50mM MES buffer (pH5.0) formula (250ml):

[0066]

[0067] 2×Tm hybridization buffer

[0068]

[0069] Store at 4°C after filtration.

[0070] ExoSAP-IT kit was purchased from US USB Company.

[0071] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0072] 1. Sample DNA extraction:

[0073] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0074] 2. PCR amplification of samples to be tested

[0075] Three pairs of primers were designed, and multiplex PCR amplified three target sequences containing three common genotypes T116C, A212G and A117G of the TGM5 gene in one step. The product sizes were 341bp, 411bp and 320bp respectively. ) are shown in Table 3 above.

[0076] Fi...

Embodiment 3

[0117] The liquid phase chip of embodiment 3 different ASPE primers detects the TGM5 gene SNP site

[0118] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0119] Taking the TGM5 gene T116C, A212G and A117G 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 T116C, A212G and A117G, and the Tag sequence at the 5' end of the ASPE primer was It is selected from SEQ ID NO.1-SEQ ID NO.6, and correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.13-SEQ ID NO.18. The specific design is shown in the following table (Table 7). 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.

[0120] Table 7 Design of liquid phase chip prep...

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Abstract

The invention discloses a TGM5 gene mutation detection liquid phase chip and a specific primer. The liquid phase chip mainly comprises: an ASPE primer composed of a tag sequence at 5'-terminal and specific primer sequences against the mutation site of a target gene at 3'-terminal, wherein the specific primer sequences comprise SEQ ID NO.7 and SEQ ID NO.8 against a T116C site, SEQ ID NO.9 and SEQ ID NO.10 against an A212G site, and / or SEQ ID NO.11 and SEQ ID NO.12 against an A117G site; a microsphere coated with an anti-tag sequence; and an amplimer. The coincidence rate between the detection result of the detection liquid phase chip and a sequencing method is 100%, and the wild and mutant parallel detection of a plurality of mutation sites can be realized.

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 TGM5 gene mutation detection and a liquid phase chip. Background technique [0002] TGM5 is called transglutaminase (Transglutaminase 5). It is located on the long arm of chromosome 15q15.2. More precisely, the base of the TGM5 gene is located between 43524792 and 43559054 base pairs on chromosome 15, including 13 exons and 12 introns. The TGM5 gene is a member of the transglutaminase family, which can promote the formation of protein cross-links between glutamine and lysine residues, thereby stabilizing protein assembly. This reaction is dependent on calcium. In this Mutations in the gene are associated with peeling acral syndrome. [0003] At present, the TGM5 gene mutation detection methods mainly include: Illumina fiber optic microbead chip technology, although Illumina fiber optic microbead chip technology is a h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C40B40/06
Inventor 许嘉森吴诗扬
Owner SUREXAM BIO TECH
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