Novel methylation library construction method and application thereof

A methylation and genome technology, applied in the field of molecular biology, can solve problems such as decreased link efficiency, expensive enzymes, and no linking ability

Inactive Publication Date: 2018-06-15
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0020] The method of self-ligation into a circle requires the use of circligase. First, the enzyme will preferentially perform intramolecular circularization, so if it is necessary to access adapters for pre-amplification, the relative linking efficiency will be greatly reduced.
Second, the enzyme is sequence-selective for the ligation of target molecules. It has no ligation ability for molecules with C at the 3' end, and the ligation efficiency for molecules with A\T\C at the 5' end is much lower than that of 5' Molecules that end in a G, so there is a severe bias
Third, the enzyme is expensive

Method used

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  • Novel methylation library construction method and application thereof
  • Novel methylation library construction method and application thereof
  • Novel methylation library construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0225] 1. Test preparation

[0226] (1) Synthesis of cohesive end adapters to be used in the method of methylation library construction:

[0227] The sticky end adapter is a double-stranded linker with a sticky end, one end of the sticky end adapter is a first sticky end, and the first sticky end includes at least a random sequence.

[0228] 1. As an example, use Primer1 and Primer2 to synthesize sticky end adapters,

[0229] Primer1: 5'-AGATCGGAAG-3'-bioten (SEQ ID NO.1);

[0230] Primer2: 5'-CTTCCGATCTNNNNNN-3' (SEQ ID NO.2); wherein, NNNNNN represents a random sequence, usually the length of the random sequence is 2-30nt, preferably 4-20nt, more preferably 5-10nt .

[0231] 2. Add the configuration reaction solution according to Table 1:

[0232] Table 1

[0233] Reagent

Volume (ul)

Primer1 (10μM)

1

Primer2 (10μM)

1

TE buffer

7

5M NaCl

1

total capacity

10

[0234] The TE buffer used in the reaction ca...

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Abstract

The invention relates to the technical field of molecular biology and in particular relates to a novel methylation library construction method and an application thereof. The novel methylation libraryconstruction method provided by the invention is suitable for single-strand connection, amplification or library construction of a DNA sample treated by using bisulfite. Due to the adoption of a method for bisulfite treatment and then adhesive end joint connection, sample information loss caused by DNA breakage resulted from drug treatment is greatly reduced.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a novel methylation library construction method and its application. Background technique [0002] Methylation is one of the most common epigenetic modification methods, which regulate gene expression without changing the nucleic acid sequence. DNA methylation usually refers to the addition of a methyl group to the fifth carbon atom of cytosine under the mediation of DNA methyltransferase to make it into 5-methylcytosine (5-mC ). Methylation modification plays an important role in gene imprinting, gene expression regulation, X chromosome inactivation, cancer occurrence and development, etc. [0003] Abnormal DNA methylation can participate in the formation of tumors by affecting the expression of proto-oncogenes and tumor suppressor genes. When tumors occur, tumor suppressor genes usually undergo overall hypermethylation, especially in the CpG island region, thereby ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N15/10C12Q1/6869C12Q1/6858
CPCC12N15/1093C12Q1/6858C12Q1/6869C40B50/06C12Q2523/125C12Q2525/191C12Q2521/501
Inventor 陈昌岳李静王芳闫丽郑冠涛路远胡秋萍张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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