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A multi-sample multi-fragment DNA methylation high-throughput sequencing method

A methylation, multi-fragment technology, applied in recombinant DNA technology, biochemical equipment and methods, DNA/RNA fragments, etc., can solve PCR recombination between alleles, cannot detect methylation status, and is time-consuming and expensive. and other problems, to achieve the effect of utilizing sequencing throughput, low incidence of PCR recombination, and sufficient sequencing throughput

Active Publication Date: 2018-04-27
上海昂朴生物科技有限公司 +2
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Problems solved by technology

[0005] The bisulfite clone sequencing method is a method with high reliability and accuracy, which can clarify the methylation status of each CpG site in the target fragment, but the disadvantage is that at least 10 clones must be sequenced to obtain Reliable data, the process is cumbersome, time-consuming and expensive
[0006] The disadvantage of BSAS is that after the PCR product is digested with a transposase, the methylation status of the sequence cut off by the enzyme at both ends of the PCR product cannot be detected; each sample needs to construct a library
Moreover, there are reports in the literature that PCR recombination between alleles occurs in amplicon sequencing

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[0029] The main technical process of the present invention: extract genomic DNA, then perform bisulfite treatment on genomic DNA, and then perform PCR amplification on the processed genomic DNA, wherein different PCR primers are used for different gene fragments, and the same gene fragments use The label (barcode) sequence distinguishes different samples, and then mixes different PCR products. The mixed PCR products are prepared for sequencing library, and then the library is subjected to QPCR quality inspection and quantification, then sequenced on the machine, and finally different sample specificity High-precision analysis of fragment methylation status.

[0030] According to an embodiment of the present invention, the sample genomic DNA can be derived from any species, including but not limited to genomic DNA of animals, plants, and insects.

[0031] According to an embodiment of the present invention, the extracted genomic DNA is treated with bisulfite, such as using EZ D...

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Abstract

A multi-sample multi-fragment DNA methylation high-throughput sequencing method of the present invention can simultaneously analyze the DNA methylation status of multiple samples and multiple target fragments in one library, the fragment length can reach about 500bp, and PCR The incidence of recombination is low, and this method also has the advantages of small initial sample size, strong pertinence, low cost, and full utilization of sequencing throughput.

Description

technical field [0001] The invention relates to the field of high-throughput genome methylation DNA detection, in particular to a multi-sample multi-segment DNA methylation high-throughput sequencing method. Background technique [0002] DNA methylation is an important epigenetic regulator. In mammalian cells, DNA methylation usually occurs on the cytosine of a CpG dinucleotide, forming 5-methylcytosine. The change of DNA methylation, especially the hypermethylation of tumor suppressor gene promoter region plays an important role in the formation and development of various tumors, while DNA hypomethylation is also related to the overexpression of oncogenes and the Instability related. [0003] Currently, methods for studying DNA methylation include microarray analysis following methylated DNA immunoprecipitation (MeMeDIP-chip) for genome-wide methylation detection, sequencing analysis following Methylated DNAimmunoprecipitation (MeDIP-seq), reduced representation bisulfit...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C40B40/06C12N15/11
Inventor 陈静王曦路
Owner 上海昂朴生物科技有限公司
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