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Construction method and detection method of trace fragmented DNA methylation detection library

A construction method and methylation technology, which can be used in chemical libraries, biochemical equipment and methods, and microbial determination/inspection. It can solve problems such as poor targeting, high sequencing costs, and inability to detect trace DNA.

Inactive Publication Date: 2018-04-20
GENETALKS BIO TECH CHANGSHA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The site is not specific, the cost of sequencing is high, and the amount of sample DNA is required to be large enough to detect the trace amount of DNA obtained in liquid biopsy

Method used

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  • Construction method and detection method of trace fragmented DNA methylation detection library
  • Construction method and detection method of trace fragmented DNA methylation detection library
  • Construction method and detection method of trace fragmented DNA methylation detection library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The region to be detected in this embodiment is located near the promoter region or the first exon region of the SEPT9 gene. The specific sequence is as follows: AGCCAGGGGGCCTAGGGGCTCCTCCGGCGGCTAGCTCTGCACTGCAGGAGCGCGGGCGCGGCGCCCCAGCCAGCGCGCAGGGCCCGGGCCCCGCCGGGGGCGCTTCCTCGCCGCTGCCCTCCGCGCGACCCGCTGCCCACCAGCCATCAT GTCGGACCCCGCGGTCAACGCG CAGCTGGATGGGATCATTTCGGACTTCGAAGGTGGGTGCTGGGCTGGCTGCTGCGGCCGCGGACGTGCTGGAGAGGACCCTGCGGGTGGGCCTGGCGCGGGACGGGGGTGCGCTGAGGGGAGACGGGAGTGCGCTGAGGGGAGACGGGAC (SEQ ID NO: 1).

[0043] 1. Design specific primers for the above regions:

[0044] SPT9-S:

[0045] ACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCGAAATCCGAAATAATCCCATCCAACTA (SEQ ID NO: 2).

[0046] 2. Design and manufacture the linker sequence of the illumina sequencing platform:

[0047] ADT-S: GATCGGAAGAGC (SEQ ID NO: 3).

[0048] All cytosine Cs in the linker sequence are methylated.

[0049] ADT-AS:

[0050] CAAGCAGAAGACGGCATACGAGATNNNNNNNIIIIIIIGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT (SEQ ID N...

Embodiment 2

[0096] 1. The specific sequence of the area to be detected in this embodiment is as follows:

[0097]CpG island region of SEPT9 gene:

[0098] AGCCAGGGGGCCTAGGGGCTCCTCCGGCGGCTAGCTCTGCACTGCAGGAGCGCGGGCGCGGCGCCCCAGCCAGCGCGCAGGGCCCGGGCCCCGCCGGGGGCGCTTCCTCGCCGCTGCCCTCCGCGCGACCCGCTGCCCACCAGCCATCATGTCGGACCCCGCGGTCAACGCGCAGCTGGATGGGATCATTTCGGACTTCGAAGGTGGGTGCTGGGCTGGCTGCTGCGGCCGCGGACGTGCTGGAGAGGACCCTGCGGGTGGGCCTGGCGCGGGACGGGGGTGCGCTGAGGGGAGACGGGAGTGCGCTGAGGGGAGACGGGAC(SEQ ID NO:7)。

[0099] CpG island region of NDRG4 gene:

[0100] TCTAAGGTTCCCCTTGGGAGTCTAAACAAAGACTACGGCAGCGCCGTCCCCTCCCCCGGGAACCCGACGCCGCGCGGCCACAGGGGGCCTGGAGGGGCGGGCAGGGCCTCGCAGCGCACCCAGCACAGTCCGCGCGGCGGAGCGGGTGAGAAGTCGGCGGGGGCGCGGATCGACCGGGGTGTCCCCCAGGCTCCGCGTCGCGGTCCCCGCTCGCCCTCCCGCCCGCCCACCGGGCACCCCAGCCGCGCAGAAGGCGGAAGCCACGCGCGAGGGACCGCGGTCCGTCCGGGACTAGCCCCAGGCCCGGCACCGCCCCGCGGGCCGAGCGCCCACACCCGCCAAACCCACGCGGGCACGCCCCCGCGGCGCACCGCCCCCAGCCCGGCCTCCGCCCCTGCAGCCGCGGGCACGCGGAGGGGCTCCTGGCTGCCCGCACCTGCACCCGCGCGTCGGCGGC(SE...

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Abstract

The invention discloses a construction method and detection method of a trace fragmented DNA methylation detection library. The library construction method of the present invention comprises: linkingfragmented DNA to a methylation linker, wherein all cytosines on the methylation linker are methylated cytosines, the methylation linker includes a long chain and a short chain which are complementaryto form a double chain, and the long chain includes a sequencing platform universal sequence, a single molecule tag sequence, a sample tag sequence and a sequencing primer binding sequence; and the DNA linked with the methylation linker is treated with sodium bisulphate, so that the cytosine C in a non-methylated CpG island is converted into uracil U; and a specific primer and a universal primerare used for PCR amplification enrichment of a target region subjected to sodium bisulfite treatment and containing a CpG site to be detected, so that a library available for on-machine sequencing isobtained. According to the invention, methylation of trace DNA taken through liquid biopsy can be detected, and the CpG island methylation status of multiple regulatory regions of multiple tumor suppressor genes can be quantitatively detected simultaneously.

Description

technical field [0001] The invention relates to the technical field of DNA methylation detection, in particular to a construction method and detection method of a micro-fragmented DNA methylation detection library. Background technique [0002] The research scope of epigenetics mainly includes DNA methylation, histone modification, chromatin reorganization, etc. DNA methylation refers to the 5-cytosine methylation (5-mC) of genomic DNA. It is a stable and widespread epigenetic modification in mammals, and it is also the most thoroughly studied epigenetic modification. genetically modified form. Under the action of DNA methyltransferase, the 5' carbon position of cytosine of genomic CpG dinucleotide is covalently bonded with a methyl group. Under normal circumstances, the CpG dinucleotides of the "junk" sequences in the human genome are relatively rare and are always in a methylated state; in contrast, the human genome is about 100-1000bp in size and rich in CpG dinucleotid...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/6869
CPCC12Q1/6869C40B50/06C12Q2535/122C12Q2525/191C12Q2523/125
Inventor 宋卓王永利
Owner GENETALKS BIO TECH CHANGSHA CO LTD
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