rad51l1 gene mutation detection specific primers and liquid phase chip

A technology of RAD51L1 and detection solution, which is applied in the field of molecular biology, can solve the problems of easy contamination of samples, narrow analysis range, and low degree of automation, and achieve the effect of avoiding uncertain factors, avoiding cross-reaction, and consistent detection results

Active Publication Date: 2016-06-22
SUREXAM BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the chromosomal variation of RAD51L1 gene is associated with the occurrence of pulmonary chondroid hamartoma and uterine leiomyoma, and RAD51L1 gene polymorphism can increase the risk of breast cancer
[0003] At present, RAD51L1 gene mutation detection methods mainly include: Illumina fiber optic bead chip technology, microarray technology and fluorescence quantitative PCR technology. Although Illumina fiber optic bead 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, which are difficult to meet the needs of practical applications. Microarray technology has problems such as high cost, complexity, low detection sensitivity, poor repeatability, narrow analysis range, and complex synthesis and fixation of probes. In addition, due to hybridization Located on the surface of the solid phase, with a certain degree of steric hindrance
Fluorescent quantitative PCR technology has the characteristics of high sensitivity, strong specificity, and high degree of automation, but it has the disadvantages of easy sample contamination and high false positive rate, and can only detect one mutation type at a time, which also cannot meet the needs of practical applications.

Method used

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  • rad51l1 gene mutation detection specific primers and liquid phase chip
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  • rad51l1 gene mutation detection specific primers and liquid phase chip

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1 RAD51L1 gene mutation detection liquid chip mainly includes:

[0034] 1. ASPE Primers

[0035] Specific primer sequences were designed for the wild-type and mutant types of the four common genotypes C167T, A76T, T187G and T99G of the RAD51L1 gene. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0036] Table 1 ASPE primer sequence of RAD51L1 gene (tag sequence + specific primer sequence)

[0037]

[0038]

[0039] 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 / LTrisBuffer.

[0040] 2. Microspheres coated with anti...

Embodiment 2

[0053] Example 2 Detection of samples using the RAD51L1 gene mutation detection liquid chip described in Example 1

[0054] The formula of described various solutions is as follows:

[0055] 50mM MES buffer (pH5.0) formula (250ml):

[0056]

[0057] 2×Tm hybridization buffer

[0058]

[0059] Store at 4°C after filtration.

[0060] ExoSAP-IT kit was purchased from US USB Company.

[0061] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0062] 1. Sample DNA extraction:

[0063] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0064] 2. PCR amplification of samples to be tested

[0065] Four pairs of primers were designed, and multiplex PCR amplified four target sequences containing four common genotypes C167T, A76T, T187G, and T99G of the RAD51L1 gene in one step. The product sizes were 359bp, 250bp, 320bp, and 290bp, respectively. 25-32) are shown in Tabl...

Embodiment 3

[0108] The liquid phase chip of embodiment 3 different ASPE primers detects the RAD51L1 gene SNP site

[0109] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0110] Taking RAD51L1 gene C167T, A76T, T187G and T99G 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 C167T, A76T, T187G and T99G, respectively, and the ASPE primer 5 The Tag sequence at the 'end is selected from SEQIDNO.1-SEQIDNO.8, and correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQIDNO.17-SEQIDNO.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.

[0111] Table 7 Design of liquid phase c...

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Abstract

The invention discloses a liquid phase chip and a specific primer for detecting RAD51L1 gene mutation. The liquid phase chip mainly comprises: ASPE primers, microspheres coated by different anti-tag sequences, and an amplification primer, wherein each ASPE primer is composed of a tag sequence at a 5' end and specific primer sequences at a 3' end and aiming at target gene mutation sites, and the specific primer sequences are as follows: SEQ ID NO.9 and SEQ ID NO.10 aiming at a C167T site, SEQ ID NO.11 and SEQ ID NO.12 aiming at an A76T site, SEQ ID NO.13 and SEQ ID NO.14 aiming at a T187G site, and / or SEQ ID NO.15 and SEQ ID NO.16 aiming at a T99G site. The coincidence rate of the detection result of the detection liquid phase chip disclosed by the invention with that of a sequencing method reaches as high as 100%, and parallel detection of a wild type and a mutant 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 RAD51L1 gene mutation detection specific primer and a liquid phase chip. Background technique [0002] RAD51-like 1 (RAD51-like1, RAD51L1), also known as hREC2 or RAD51B, is located on the long arm of chromosome 14 14q23-24, specifically between 68286495 and 69062737 base pairs on chromosome 14, and the RAD51L1 gene encodes the repair double strand An enzyme that breaks DNA, but overexpression of this gene leads to cell cycle G1 delay and apoptosis, suggesting a role for this protein in DNA damage. The protein encoded by RAD51L1 is a member of the Rad51 protein family. RAD51 family members are indispensable conserved proteins for homologous recombination DNA repair, and participate in the homologous recombination repair pathway of double-strand DNA breaks during DNA replication. In addition, the chromosomal variation of RAD51L1 gene is a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B40/06C12Q1/68C12N15/11
Inventor 陈昌华许昌有
Owner SUREXAM BIO TECH
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