XRCC2 gene mutation detection specific primers and liquid chip

A detection solution and specificity technology, applied in the field of molecular biology, can solve the problems of easy contamination of samples, complicated operation, high false positive rate, etc., and achieve the effect of avoiding uncertain factors, low cross-reaction rate and avoiding cross-reaction

Active Publication Date: 2014-06-18
SUREXAM BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the XRCC2 gene mutation detection methods mainly include: fluorescent quantitative PCR technology, SNPlexTM System technology and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Fluorescent quantitative PCR technology has high sensitivity, strong specificity, and high degree of automation. However, it has the disadvantages of easy sample contamination and high false positive rate, and can only detect one mutation type at a time, which cannot meet the needs of practical applications.
The SNPlexTM System technology has high requirements on the specificity of SNP sequences, and it cannot randomly analyze the selected single nucleotide polymorphism sites, so it is difficult to apply to clinical detection and diagnosis. Moreover, the method is complicated to operate and cannot meet the needs of practical applications.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a soft ionization technology that has powerful and mature functions in the detection of protein and other biological macromolecules. However, in the field of nucleic acid detection, due to the particularity of nucleic acid molecules, detection is subject to certain limit

Method used

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  • XRCC2 gene mutation detection specific primers and liquid chip
  • XRCC2 gene mutation detection specific primers and liquid chip
  • XRCC2 gene mutation detection specific primers and liquid chip

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1 XRCC2 gene mutation detection liquid chip mainly includes:

[0031] 1. ASPE Primers

[0032] Specific primer sequences were designed for wild-type and mutant types of four common genotypes of XRCC2 gene, G87A, G158C, A82G and A155C. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0033] Table 1 ASPE primer sequence of XRCC2 gene (tag sequence + specific primer sequence)

[0034]

[0035]

[0036] 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.

[0037] 2. Microspheres coated with anti-tag sequences

...

Embodiment 2

[0049] Example 2 Detection of samples using the XRCC2 gene mutation detection liquid chip described in Example 1

[0050] The formula of described various solutions is as follows:

[0051] 50mM MES buffer (pH5.0) formula (250ml):

[0052]

[0053] 2×Tm hybridization buffer

[0054]

[0055] Store at 4°C after filtration.

[0056] ExoSAP-IT kit was purchased from US USB Company.

[0057] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0058] 1. Sample DNA extraction:

[0059] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0060] 2. PCR amplification of samples to be tested

[0061] 4 pairs of primers were designed, and multiplex PCR amplified 4 target sequences containing four common genotypes G87A and G158C, A82G and A155C of the XRCC2 gene in one step. The product sizes were 239bp, 397bp, 179bp and 317bp respectively. NO.25-32) see Table 3 above.

[0...

Embodiment 3

[0105] The liquid phase chip of embodiment 3 different ASPE primers detects the SNP site of XRCC2 gene

[0106] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0107] Taking the XRCC2 gene G87A, G158C, A82G and A155C 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 G87A, G158C, A82G and A155C, 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.

[0108] Table 7 Design of liquid...

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Abstract

The invention discloses a XRCC2 gene mutation detection liquid chip, and specific primers. The XRCC2 gene mutation detection liquid chip mainly comprises: ASPE primers, wherein each ASPE primer is composed of 5'-terminal tag sequence, and 3'-terminal specific primer sequence targeting target gene mutation sites, and the specific primer sequence comprises SEQ ID No.9 and SEQ ID No.10 targeting G87A site, SEQ ID No.11 and SEQ ID No.12 targeting G158C site, SEQ ID No.13 and SEQ ID No.14 targeting A82G site, and/or SEQ ID No.15 and SEQ ID No.16 targeting A155C site; microballoons coated with anti-tag sequences; and amplification primers. Self-agreement ratio of detection results of the liquid chip with detection results of sequencing is as high as 100%; and parallel detection of wild types and mutant types of a plurality of mutation sites is 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 detecting XRCC2 gene mutation and a liquid phase chip. Background technique [0002] X-ray repair complementing defective repair in Chinese hamster cells 2 (XRCC2), located on chromosome 7 7q36.1. The XRCC2 gene is a member of the RecA / Rad51-related protein family and is involved in homologous recombination to maintain chromosome stability and repair DNA damage. The XRCC2 gene is an important gene involved in the repair of DNA double-strand breaks, and plays an important role in homologous recombination repair (homology recombination, HR) and non-homology end joining (non-homology endjoining, NHEJ). The resulting individual differences in repair ability may be an important factor in determining differences in tumor risk. XRCC2 gene polymorphisms are associated with tumor susceptibility to colorectal cancer, bladder ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6827C12Q1/686C12Q2563/149C12Q2531/113
Inventor 陈昌华胡文晖
Owner SUREXAM BIO TECH
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