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Method for constructing DNA (deoxyribonucleic acid) methylation library and application of method

A technology of methylation and DNA molecules, applied in chemical libraries, biochemical equipment and methods, combinatorial chemistry, etc., can solve the problem of methylation in other regions such as CHH and CHG regions, which may also be related to tumor occurrence and cannot be detected Detect and other problems to achieve the effect of avoiding template loss, saving time, and simple operation

Active Publication Date: 2020-02-07
SHENZHEN HUADA GENE INST
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Problems solved by technology

However, its disadvantage is that it can only detect highly methylated modified CpG sites, and the methylated modifications of low methylated modified CpG sites or CHG and CHH sites cannot be detected
Abnormal DNA methylation is closely related to the occurrence and development of tumors, but not only the highly methylated CpG region is related to the occurrence and development of tumors, the methylation of hypomethylated modified regions and other regions such as CHH and CHG regions It may also be closely related to the occurrence and development of tumors
[0007] However, the detection method for DNA methylation needs to be further improved

Method used

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  • Method for constructing DNA (deoxyribonucleic acid) methylation library and application of method
  • Method for constructing DNA (deoxyribonucleic acid) methylation library and application of method
  • Method for constructing DNA (deoxyribonucleic acid) methylation library and application of method

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[0050] According to another specific embodiment of the present invention, the method for constructing a DNA methylation library further includes; performing PCR amplification on the DNA molecule connected by the linker sequence, so as to obtain a PCR amplification product; The amplification product is denatured into single-stranded DNA, and the single-stranded DNA is circularized into circular single-stranded DNA. According to an embodiment of the present invention, the amplification product is denatured into a single-stranded DNA by high temperature, and the single-stranded DNA is circularized under the action of a circularization primer, T4DNA Ligase (T4DNA Ligase) and ATP to obtain a circularized product. The circularization product can be subjected to the action of exo I and exo III to obtain a single-stranded circular DNA library that can be sequenced on the machine. Preferably, the PCR amplification product is purified by magnetic beads, and then denatured into a single ...

Embodiment 1

[0058] In this embodiment, circulating tumor DNA is obtained by using the blood samples of tumor patients, and then bisulfite-converted single-stranded DNA molecules are obtained by using bisulfite conversion treatment; and through linear amplification, single-stranded DNA molecules are respectively Linker sequences are connected to both ends of the adapter sequence; PCR amplification is performed using the DNA molecule connected by the adapter sequence to obtain a PCR amplification product; the PCR amplification product is denatured into single-stranded DNA and circularized into circular single-stranded DNA. Use the BGISEQ sequencing platform to obtain the sequencing results, and obtain the DNA methylation detection results of tumor patients.

[0059] At the same time, the detection results were compared with the methylation detection results of the tumor tissues of the above-mentioned tumor patients by using the method of routine whole genome library construction and sequenci...

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Abstract

The invention relates to the field of gene sequencing and in particular relates to a method for constructing a DNA (deoxyribonucleic acid) methylation library and application of the method. The methodcomprises the following steps: performing bisulfite conversion treatment on a DNA sample so as to obtain single-chain DNA molecules after bisulfite conversion; performing linear amplification, and respectively connecting a first joint sequence and a second joint sequence with both ends of the single-chain DNA molecules so as to obtain DNA molecules connected with the joint sequences; and constructing the DNA methylation library by using the DNA molecules connected with the joint sequences. The library provided by the invention is adopted for sequencing and analysis, whole-genome methylation detection of a small amount of DNA can be achieved, and compared with a conventional technique, the method is wide and comprehensive in cover range and in addition has the advantages of being simple inexperiment operation and short in cycle.

Description

technical field [0001] The present invention relates to the field of gene sequencing, in particular to a method for constructing a DNA methylation library and its application, in particular to a method for constructing a DNA methylation library, a DNA methylation detection method and a DNA methylation detection kit and its uses. Background technique [0002] Circulating tumor DNA (ctDNA) refers to the DNA released by tumor cell necrosis and apoptosis into the plasma, and degrades the body's endogenous DNA in the circulating blood. Circulating nucleic acids were first discovered by Mandel and Metais in 1947, but due to the lack of highly sensitive and specific experimental methods, the research on the correlation between free DNA in blood and diseases has progressed slowly in a long period of time. Until the emergence of effective separation of free DNA technology, the application of detection technology combined with special fluorescent dyes and PCR technology has made the ...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6858
CPCC40B50/06C12Q1/6858C12Q2523/125C12Q2535/122C12Q2525/191
Inventor 罗慧娟谢颖易吉于源于竞吴逵李南南陈小芳
Owner SHENZHEN HUADA GENE INST
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