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Method for simultaneously detecting genomic DNA polymorphism and methylation

A DNA polymorphism and genome technology, applied in the field of molecular biology, can solve the problems of time-consuming and laborious, unable to meet the direct connection and other problems

Pending Publication Date: 2019-04-02
SHANGHAI EPIPROBE BIOTECH CO LTD
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Problems solved by technology

However, current research methods can only measure genomic variation and epigenome features independently, which is not only time-consuming and laborious, but also can only be compared indirectly when studying the interaction between genome and epigenome, which cannot meet the requirements of both. The need for a direct link between

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  • Method for simultaneously detecting genomic DNA polymorphism and methylation
  • Method for simultaneously detecting genomic DNA polymorphism and methylation
  • Method for simultaneously detecting genomic DNA polymorphism and methylation

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Embodiment 1

[0026] Example 1 The method for simultaneously performing genomic DNA polymorphism and methylation detection

[0027] The technical route of the present invention is as figure 1 As shown, the specific method is:

[0028] 1) DNA fragmentation

[0029] Take 3 μg of genomic DNA from 97L liver cancer cells (MHCC-97L cells, purchased from Shanghai Bangjing Industrial Co., Ltd.), add water to adjust the volume of DNA to 85 μL, and use a Biorupter to disrupt DNA at 4°C; power: 80%, working time 30s , the gap time is 30s, 6 cycles, the DNA fragment is interrupted to about 300bp (such as figure 2 , where ultrasonic fragmentation of genomic DNA to a major band of 300 bp size can be seen).

[0030] 2) 3' end enzyme digestion treatment

[0031] 1. Prepare the terminal repair system by operating on ice, and add in the following order:

[0032] dna

35μL

10×NEB Buffer2 (purchased from NEB, item number catB7002)

10 μL

Klenow

5μL

wxya 2 o

...

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Abstract

The invention provides a method for simultaneously detecting genomic DNA polymorphism and methylation. The method comprises the following steps: performing DNA fragmentation, and digesting the 3' endof the DNA fragment with DNA exonuclease; adding a dNTPs mixture containing 5-hydroxymethylcytosine deoxyribonucleotide, and performing completion the 3' end of the DNA fragment; performing bisulfiteconversion treatment on the modified DNA fragment and connecting an adaptor adapted to a high-throughput sequencing platform, and finally constructing a sequencing library by PCR amplification. By combining a double-end sequencing mode of the high-throughput sequencing platform, genomic DNA polymorphism and methylation detection can be performed simultaneously, and the goal of simultaneous detection of genome and epigenome is achieved.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for simultaneously detecting genomic DNA polymorphism and methylation. Background technique [0002] The genome is the carrier for storing human genetic information. A large number of scientific studies have confirmed the close relationship between genomic variations such as single base mutations, DNA fragment rearrangements, insertions, deletions, and copy number abnormalities and various human diseases. The completion of the Human Genome Project (HGP) has rapidly increased our understanding of the relationship between various forms of human genetic variation and their evolutionary history and various disease phenotypes. Medical plans are benefiting more and more patients. Molecular targeted drugs developed for specific types of genomic abnormalities have made human beings fight against diseases. It can be said that the accurate interpretation of genetic inf...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C40B40/06
CPCC12Q1/6869C40B40/06C12Q2535/122C12Q2525/191
Inventor 李振艳罗怀兵
Owner SHANGHAI EPIPROBE BIOTECH CO LTD
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