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Primer pair and kit for detecting P53 gene epigenetic modification difference in peripheral blood free DNA

An epigenetics, peripheral blood technology, applied in recombinant DNA technology, DNA/RNA fragment, determination/inspection of microorganisms, etc., can solve the problems of unclear epigenetic modification function and little attention

Active Publication Date: 2018-10-19
朱运峰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is known that the methylation of gene promoters is negatively correlated with gene expression (affecting the binding of transcription factors and RNA polymerase), but the function of epigenetic modifications in gene bodies is still unclear, and current research mainly focuses on the Epigenetic modifications in gene promoter regions, while little attention has been paid to changes in various epigenetic modifications in the gene body

Method used

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  • Primer pair and kit for detecting P53 gene epigenetic modification difference in peripheral blood free DNA
  • Primer pair and kit for detecting P53 gene epigenetic modification difference in peripheral blood free DNA
  • Primer pair and kit for detecting P53 gene epigenetic modification difference in peripheral blood free DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Primer pair set for detecting the difference in epigenetic modification of P53 gene in peripheral blood cell-free DNA

[0048] According to the sequences of different segments of the P53 gene, PCR primers were designed and a large number of screening experiments were carried out, and finally a set of primer pairs with strong specificity and high sensitivity for detecting the difference in epigenetic modification of the P53 gene in peripheral blood cell-free DNA was obtained. , the primer pair set consists of the nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10 in the sequence listing;

[0049] Among them, the sequences of SEQ ID No.1 and SEQ ID No.2 are respectively the upstream primer and the downstream primer for amplifying the exon 3 region of the P53 gene with a length of 140bp (P53-E3-140), and the sequence SEQ ID No.3 is A probe for amplifying the fragment of this region; SEQ ID No.4 and SEQ ID No.5 are respectively the upstream primer and the d...

Embodiment 2

[0078] Example 2 Kit for detecting the difference in epigenetic modification of P53 gene in cell-free DNA of peripheral blood

[0079] In peripheral blood cell-free DNA, the kit for detecting the difference in epigenetic modification of P53 gene includes the primers in Example 1 consisting of the nucleotide sequences shown in SEQ ID No. 1 to SEQ ID No. 10 of the Sequence Listing Pair group, PCR reaction solution (Premix Ex Taq TM ), QC control DNA samples and ddH 2 O;

[0080] The quality control control DNA sample is naked DNA, for example, it can be a PCR product without epigenetic modification; further, in order to better simulate the concentration of free DNA, the PCR product needs to be highly diluted to make it equal to the sample concentration. Re-amplification as a template; wherein, the quality control control DNA sample includes a 140bp naked DNA fragment in the exon 3 region of the P53 gene, a 204 bp naked DNA in the P53 gene exon 3 region, and a P53 gene exon 4 r...

Embodiment 3

[0081] Embodiment 3 Detection method and process

[0082] 1) Extract cell-free DNA from peripheral blood;

[0083] 2) Using a primer pair set consisting of the nucleotide sequences shown in SEQ ID No. 1 to SEQ ID No. 10 in the sequence table, the peripheral blood free DNA and quality control control DNA samples were denatured at high temperature and low temperature respectively. Real-time fluorescence quantitative PCR amplification under conditions,

[0084] The qPCR reaction system of each primer set of the reaction body is shown in Table 1-4:

[0085] Table 1. x1 sequence qPCR reaction system

[0086]

[0087] Table 2. x2 sequence qPCR reaction system

[0088]

[0089]

[0090] Table 3. x3 sequence qPCR reaction system

[0091]

[0092] Table 4. x4 sequence qPCR reaction system

[0093]

[0094] The reaction conditions are:

[0095] High Temperature (HT): Pre-denaturation at 95℃ for 5min; 94℃ for 5s, 60℃(x1,x3) / 58℃(x2,x4) for 15 seconds, 72℃ for 30s, 50 ...

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Abstract

The invention discloses a primer pair and a kit for detecting P53 gene epigenetic modification difference in peripheral blood free DNA. The primer pair for detecting P53 gene epigenetic modification difference in peripheral blood free DNA disclosed by the invention is prepared from nucleotide sequences shown from SEQ ID No.1 to SEQ ID No.10 of a sequence table. The invention further discloses thekit containing the primer pair. According to the primer pair and the kit for detecting P53 gene epigenetic modification difference in the peripheral blood free DNA disclosed by the invention, the detection has high sensitivity and specificity and has important value in tumor risk prediction and curative effect evaluation.

Description

technical field [0001] The present invention relates to the field of biotechnology. More specifically, it relates to a primer pair set and a kit for detecting the difference in epigenetic modification of P53 gene in cell-free DNA of peripheral blood. Background technique [0002] In the clinical needs of tumor prevention and treatment, liquid biopsy has become an important means of tumor risk prediction, targeted drug administration guidance, and efficacy evaluation. Early risk assessment of tumors is the most important part of tumor prevention. Blood is undoubtedly the best sample for liquid testing because it contains a lot of tumor-related information. Among them, cell-free DNA in peripheral blood is a hot spot of current liquid biopsy. The cell-free DNA exists in plasma or serum, and it may come from the release of normal cell death. It may also come from the death release of tumor or precancerous cells, and both normal cells and tumor cells can actively release. [00...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6886C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2600/156C12Q2545/113C12Q2531/113C12Q2563/107
Inventor 朱运峰石焕焕操清兰刘纯希
Owner 朱运峰
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