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Kit for detecting key mutant gene of DNA cross-damage synthesis repair pathway

A mutation gene and kit technology, which is applied in the field of kits for detection of key mutation genes in DNA trans-damage synthesis and repair pathways, can solve the problems of high error possibility and mutagenic tendency

Pending Publication Date: 2021-02-12
SUZHOU HEALTH COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The DNA trans-damage synthesis repair pathway does not rely on the principle of complementary base pairing when inserting nucleotides, so the repair pathway has a high error probability and mutagenic tendency

Method used

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  • Kit for detecting key mutant gene of DNA cross-damage synthesis repair pathway
  • Kit for detecting key mutant gene of DNA cross-damage synthesis repair pathway
  • Kit for detecting key mutant gene of DNA cross-damage synthesis repair pathway

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0115] Example 1 Detection of mutations in USP1, POLH, PCNA genes

[0116] 1. Build a sample library

[0117] 1. Extraction of DNA

[0118] According to the routine DNA extraction method of tissue samples, the DNA of the samples was extracted with a kit from QIAGEN, Germany.

[0119] 2. DNA Fragmentation

[0120] Fragment the DNA sample using a DNA shredder to make the fragment length 150-200bp.

[0121] 3. DNA sample library quality inspection

[0122] Use a bioanalyzer to perform qualitative and quantitative DNA analysis to confirm that the peak length of the DNA fragment is reasonable.

[0123] 4. DNA end repair

[0124] Use T4 polymerase and Klenow E. coli polymerase fragments to blunt the DNA 5' overhanging sticky ends and 3' overhanging sticky ends to generate blunt ends for subsequent blunt end ligation. The reaction system is shown in Table 1, and the reaction was carried out in a PCR amplification instrument at 20° C. for 30 min.

[0125] 5. Add base A to the 3' ...

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Abstract

The invention provides a kit for detecting key mutant genes of a DNA cross-damage synthesis repair pathway, a probe library is composed of probes shown as SEQ ID NO.1-175, and the detection kit prepared from the probe library can be used for detecting genes related to the DNA cross-damage synthesis repair pathway. A target DNA fragment to be analyzed is enriched by using the probe library, and then the enriched DNA fragment is sequenced by a sequencing means, so that the effectiveness of gene detection is realized. The DNA cross-damage synthesis repair pathway key mutant gene can be used for predicting tumor occurrence tendency, evaluating tumor malignancy degree and clinical prognosis and guiding screening, research and development of new drugs, so that the DNA cross-damage synthesis repair pathway key mutant gene has important guiding significance in the research direction of gene functionality.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a kit for detecting key mutation genes of DNA cross-damage synthesis and repair pathways. Background technique [0002] Abnormal DNA damage repair pathways are closely related to the tendency of tumorigenesis and the sensitivity of DNA damage-induced anti-tumor drugs to cells. The DNA translesion synthesis repair pathway is one of the most important pathways in the DNA repair mechanism. The repair process is simple. When DNA encounters damage during replication and stagnates, DNA polymerase separates from helicase and generates DNA single strands at the damaged site, and RPA seals the DNA single strands. Then, the ubiquitinated ligases Rad6 and Rad18 monoubiquitinate PCNA, and the low-fidelity DNA polymerase (without proofreading function) replaces the high-fidelity polymerase, and performs nucleotide modification on the complementary strand of the damaged DNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6827
CPCC12Q1/6886C12Q1/6827C12Q2600/156C12Q2600/118C12Q2600/106C12Q2531/113C12Q2535/122
Inventor 刘松柏位伟
Owner SUZHOU HEALTH COLLEGE