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PDLIM5 gene mutation detection specific primer and liquid phase chip

A detection solution and specificity technology, applied in the field of molecular biology, can solve the problems of difficulty in meeting the needs of practical applications, low degree of automation, and many manual operations, and achieve the effects of avoiding uncertain factors, simple steps, and avoiding cross-reactions

Inactive 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 PDLIM5 gene mutation detection methods mainly include: Illumina fiber optic microbead chip technology, direct sequencing method and fluorescent quantitative PCR technology. Although Illumina fiber optic microbead chip technology is a high-throughput detection system with high sensitivity and accuracy, it has a low degree of automation. , there are many manual operations, it is difficult to meet the needs of practical applications
However, other detection techniques based on PCR, such as direct sequencing and fluorescent quantitative PCR technology, have the disadvantages of low sensitivity, easy contamination of samples, and high false positive rate, and the detection throughput of direct sequencing method and fluorescent quantitative PCR technology limitations, only one type of mutation can be detected at a time, which cannot meet the needs of practical applications

Method used

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  • PDLIM5 gene mutation detection specific primer and liquid phase chip

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1 PDLIM5 gene mutation detection liquid chip mainly includes:

[0049] 1. ASPE Primers

[0050] Specific primer sequences were designed for wild-type and mutant types of four common genotypes of PDLIM5 gene, G76A, G277T, C100T and T132C. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0051] Table 1 ASPE primer sequence of PDLIM5 gene (tag sequence + specific primer sequence)

[0052]

[0053]

[0054] Each ASPE primer consists of 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 Table 1 above). 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.

[0055] 2. Microspheres coated with anti-tag sequen...

Embodiment 2

[0066] Example 2 Detection of samples using the PDLIM5 gene mutation detection liquid chip described in Example 1

[0067] The formula of described various solutions is as follows:

[0068] 50mM MES buffer (pH5.0) formula (250ml):

[0069]

[0070] 2×Tm hybridization buffer

[0071]

[0072] Store at 4°C after filtration.

[0073] ExoSAP-IT kit was purchased from US USB Company.

[0074] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0075] 1. Sample DNA extraction:

[0076] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0077] 2. PCR amplification of samples to be tested

[0078] 4 pairs of primers were designed, and multiplex PCR amplified 4 target sequences containing four common genotypes G76A and G277T, C100T and T132C of the PDLIM5 gene in one step. The product sizes were 227bp, 395bp, 287bp and 366bp respectively. NO.25-32) see Table 3 above.

...

Embodiment 3

[0121] The liquid phase chip of embodiment 3 different ASPE primers detects the SNP site of PDLIM5 gene

[0122] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0123] Taking the PDLIM5 gene G76A, G277T, C100T and T132C 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 G76A, G277T, C100T and T132C, respectively, and the ASPE primer 5 The Tag sequence at the 'end is selected from SEQ ID NO.1-SEQ ID NO.8. Correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.17-SEQ ID NO.24. 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.

[0124] Table 7 Design of li...

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Abstract

The invention discloses a PDLIM5 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.9 and SEQ ID NO.10 against a G76A site, SEQ ID NO.11 and SEQ ID NO.12 against a G277T site, SEQ ID NO.13 and SEQ ID NO.14 against a C100T site, and SEQ ID NO.15 and SEQ ID NO.16 against a T132C 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 PDLIM5 gene mutation detection specific primer and a liquid phase chip. Background technique [0002] PDLIM5 is a LIM protein and also an Enigmahomologue LIM domain (ENH) protein, the English name is Postsynaptic Density-95 / discs large / Zone occludens-1(PDZ)and Lin-11,Isl-1,Mec-3(LIM) domain 5, located on chromosome 4 4q22.3, its composition is similar to that of Enigma protein, consisting of 1 PDZ domain and 3 LIM domains. As an adapter protein in the nervous system, ENH protein can interact with PKCβ, ε, γ, and can be phosphorylated by PKCε, β1. ENH can also bind to L / N-type calcium ion channels and affect the opening and closing of these channels. In the heart, ENH has been reported to be associated with cardiac hypertrophy, ENH mediates phosphorylation of L-type calcium channels by PKD1, and overactivation of PKD leads to cardiac hype...

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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