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Excision repair cross complementation group 2 (ERCC2) gene mutation detection specific primers and liquid chip kit

A kit and detection solution technology, applied in the field of molecular biology, can solve problems such as the use of antibodies, the lack of uniform standards for judging the results of operation steps, affecting promotion and application, and detection limitations, so as to avoid uncertain factors and improve detection accuracy , The detection effect is consistent

Active Publication Date: 2016-11-23
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 used to detect gene mutations with altered restriction sites, and can directly determine the genotype. However, this method cannot be used for the detection of gene mutations without new restriction sites.
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
Although immunohistochemistry has the advantages of specificity, strong sensitivity, and simple operation, there is no uniform standard for the use of antibodies, operating procedures, and result judgments, which affects its clinical promotion and application.

Method used

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  • Excision repair cross complementation group 2 (ERCC2) gene mutation detection specific primers and liquid chip kit
  • Excision repair cross complementation group 2 (ERCC2) gene mutation detection specific primers and liquid chip kit
  • Excision repair cross complementation group 2 (ERCC2) gene mutation detection specific primers and liquid chip kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] The ERCC2 gene mutation detection liquid chip described in this embodiment mainly includes:

[0024] 1. ASPE Primers

[0025] Specific primer sequences were designed for wild-type and mutant types of four common genotypes of ERCC2 gene, G934A, T128G, C2133T and A396C. ASPE primers consist of "tag sequence + specific primer sequence". ASPE primer sequences are shown in the table below:

[0026] ASPE primer sequence (tag sequence+specific primer sequence) of table 1 ERCC2 gene

[0027]

[0028]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). Among them, the bases marked in the "box" were used as the target to detect the wild-type and mutant bases of the mutation site, and all ASPE primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd. Each synthesized ...

Embodiment 2

[0040] Example 2 Detection of samples using the ERCC2 gene mutation detection liquid chip described in Example 1

[0041] The formula of described various solutions is as follows:

[0042] 50mM MES buffer (pH5.0) formula (250ml):

[0043]

[0044] 2×Tm hybridization buffer

[0045]

[0046] Store at 4°C after filtration.

[0047] ExoSAP-IT kit was purchased from US USB Company.

[0048] Biotin-labeled dCTP was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0049] 1. Sample DNA extraction:

[0050] Refer to the relevant methods of DNA extraction in "Molecular Cloning" to obtain the DNA to be detected.

[0051] 2. PCR amplification of samples to be tested

[0052] Four pairs of primers were designed, and multiplex PCR amplified four target sequences containing four target detection mutation sites G934A, T128G, C2133T and A396C of the ERCC2 gene in one step. The product sizes were 334bp, 284bp, 301bp and 381bp, respectively. SEQ ID NO.25...

Embodiment 3

[0094] The liquid phase chip kit of embodiment 3 different ASPE primers detects the SNP site of ERCC2 gene

[0095] 1. Design of liquid phase chip preparation (selection of Tag sequence and Anti-Tag sequence)

[0096] Taking the liquid-phase chip for detecting mutations at the G934A, T128G and C2133T sites of the ERCC2 gene as an example, the specific primer sequences at the 3' end of the ASPE primers were designed for the wild-type and mutant types of G934A, T128G and C2133T, respectively, and the Tag sequence at the 5' end of the ASPE primers It is selected from SEQ ID NO.1-SEQ ID NO.8, and correspondingly, the anti-tag sequence coated on the microsphere and complementary to the corresponding tag sequence is selected from SEQ ID NO.7-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....

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Abstract

The invention discloses an excision repair cross complementation group 2 (ERCC2) gene mutation detection liquid chip and specific primers. The liquid chip mainly comprises ASPE primers and each one of the primers comprises a tag sequence at the end 5' and a specific primer sequence at the end 3' aiming at a target gene mutation site. The specific primer sequence comprises sequences shown in the formulas of SEQ ID NO. 9 and SEQ ID NO. 10 aiming at the G934A site, sequences shown in the formulas of SEQ ID NO. 11 and SEQ ID NO. 12 aiming at the T128G site, sequences shown in the formulas of SEQ ID NO. 13 and SEQ ID NO. 14 aiming at the C2133T site, and sequences shown in the formulas of SEQ ID NO. 15 and SEQ ID NO. 16 aiming at the A396C site. The liquid chip also comprises anti-tag sequence-coated microspheres and amplification primers. The detection result of the detection liquid chip and the detection result of the sequencing method have a coincidence rate of 100%. The ERCC2 gene mutation detection liquid chip realizes individual or parallel detection of multiple wild type and mutant type sites.

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 ERCC2 gene mutation and a liquid phase chip kit. Background technique [0002] ERCC2 (excision repair cross completion group2), located at the 19q13.3 position of chromosome 19, has a total of 23 exons, the full length of the gene is 19.0kb, the full length of the mRNA is 2355bp, and it encodes a protein containing 761 amino acids. The protein encoded by ERCC2 is involved in transcription-coupled nucleotide excision repair, which recognizes large-scale lesions unrelated to repair structures, such as large adducts and thymine dimers, and is a member of the basic transcription factor complex BTF2 A component of / TFIIH. This protein has ATP-dependent DNA unwinding activity and belongs to the RAD3 / XPD helicase subfamily. Loss of function of ERCC2 results in the inability of the basic transcription factor complex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 吴诗扬廖传荣刘志明
Owner SUREXAM BIO TECH
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