Method for quantitative determination on methylation by two-nucleotide synthesis pyrosequencing

A two-nucleotide, quantitative detection technology, applied in the biological field, can solve the problems of DNA template sequence analysis that cannot be multiple methylated, exclude, limit the scope of sequence analysis, etc. Reliability, the effect of broadening the scope of analysis

Active Publication Date: 2015-12-09
SOUTHEAST UNIV
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Problems solved by technology

However, the invention patent only analyzes one DNA template sample, and cannot analyze DNA template sequences containing multiple methylations, which limits the scope of sequence analysis
Since the PCR product after bisulfite treatment needs to distinguish C and T bases, and (dATPαS+dGTP) synthetic pyrosequencing has no way to distinguish these two bases, therefore, the sequencing of two nucleotides does not include this combination form

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  • Method for quantitative determination on methylation by two-nucleotide synthesis pyrosequencing
  • Method for quantitative determination on methylation by two-nucleotide synthesis pyrosequencing
  • Method for quantitative determination on methylation by two-nucleotide synthesis pyrosequencing

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Embodiment

[0043] The method for analyzing the five methylation sites of the CpG island of the RARβ2 gene promoter in human samples, the specific methods include:

[0044] (1) Genomic DNA in peripheral blood was extracted from the blood sample using traditional protein kinase K and phenol / chloroform extraction methods;

[0045] (2) Sodium bisulfite modification of genomic DNA: Genome DNA was treated with sodium bisulfite according to the operation procedure provided by CpGenomeTurboBisulfiteModification Kit (Millipore Company), and the modified DNA was purified and recovered;

[0046] (3) PCR amplification: PCR primers: 5'-biotin-GTTGTTTGAGGATTGGGATG-3', 5'-ATACCCAAAACAAACCCTACTC-3' and 200mg of genomic DNA treated with sodium bisulfite, 0.2mMdNTP, 1UTaqDNA polymerase, 1× amplification Increasing buffer, 1.8mM MgCl 2 50μL PCR amplification system for amplification, the amplification conditions are: initial denaturation at 95°C for 5min; 40 thermal cycles: denaturation at 94°C for 30s, a...

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Abstract

The invention discloses a method for quantitative determination on methylation by two-nucleotide synthesis pyrosequencing. The method comprises the steps of performing two-nucleotide synthesis pyrosequencing on a PCR product generated by converting a gene group DNA (deoxyribonucleic acid) through a hydrosulphite, and continuously recording each sequencing reaction to acquire sequencing encoding information including types of two nucleotides and a nucleotide synthesis number thereof. Basic groups C in all CG locuses in a determined target area sequence are converted into T with different degrees, and then correlation analysis is performed by using the information acquired by two-nucleotide synthesis pyrosequencing, thus realizing quantitative determination on methylation in the target area sequence. The method can greatly improve a sequencing length, widens the analysis range of traditional pyrosequencing, and is suitable for methylation analysis of single-sample PCR products and mixed-sample PCR product sequences.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a PCR product sequence analysis method, in particular to a method for quantitatively detecting methylation by pyrosequencing two nucleotide synthesis. Background technique [0002] The development and completion of the Human Genome Project and various model organism genome projects has brought mankind into the post-gene era, which has had a huge impact on contemporary biological and medical research, and the related disciplines of molecular biology have developed rapidly. It will become possible to understand the differences of life, the law of disease occurrence and development, and the interaction between drugs and living organisms at the genetic level. As far as gene sequence analysis is concerned, the focus of the post-genome era has shifted from whole-genome sequence determination to the comparison of individual genetic differences and inter-species genetic differences in the geno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2563/131C12Q2563/143C12Q2565/301
Inventor 肖鹏峰刘文斌
Owner SOUTHEAST UNIV
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