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Targeted enrichment method for trace DNA and use thereof

A DNA-targeted and targeted technology, applied in the field of sequencing, achieves the effects of low cost, high capture efficiency, and reduced capture and library construction costs

Inactive Publication Date: 2021-10-26
徐州海纳生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This feature is just suitable for rolling circle amplification of DNA polymerase with strand displacement activity, which solves the problem of indiscriminate amplification due to random primers and achieves the purpose of targeted enrichment

Method used

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  • Targeted enrichment method for trace DNA and use thereof
  • Targeted enrichment method for trace DNA and use thereof
  • Targeted enrichment method for trace DNA and use thereof

Examples

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Effect test

Embodiment 1

[0053] In this example, a synthetic wild-type internal reference DNA sequence with 10,000 molecules of human cfDNA was incorporated, and a synthetic mutant internal reference DNA sequence was incorporated at the same time, so that the ratio of the number of molecules between the mutant type and the wild type was 0.5%. Using the method of the present invention The principle is to design mutant amplification primers for enrichment detection of mutant types, among which:

[0054] The wild-type DNA 5'→3' sequence is SEQID NO.2:

[0055] GTTGAACAATCGTCGCAATTTAGTCACAAAACAGTTACACAAACGTTGCAACGTGCGACTGTGTTCTGCTGTCGTCGCAAAAAAGTTGCACAAAGCGACTGTTTTGTGATATTTTGGAATAGGTCACAAGACAAACTTTTGAAATCCACA

[0056] Mutant DNA 5'→3' sequence is SEQID NO.3:

[0057] GTTGAACAATCGTCGCAATTTAGTCACAAAACAGTTACACAAACGTTGCAACGTGCGGCTGTGTTCTGCTGTCGTCGCAAAAAAGTTGCACAAAGCGACTGTTTTGTGATATTTTGGAATAGGTCACAAGACAAACTTTTGAAATCCACA

[0058] The stem-loop linker sequence used in this example is SEQID NO.1:

[0059] GATC...

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Abstract

The invention relates to the technical field of sequencing, in particular to a targeted enrichment method for trace DNA and use thereof. The targeted enrichment method for trace DNA comprises the following steps: S1, adding an A tail to a tail end of fragmented genome DNA; S2, connecting the fragmented DNA added with the A tail obtained in step S1 with a stem-loop joint to form annular DNA; S3, carrying out a rolling circle amplification enrichment on the annular DNA obtained in step S2 by adopting a target primer; and S4, constructing a sequencing library. The capture method can be applied to trace DNA, has high targeted enrichment efficiency and is simple and convenient to operate, and can be used for target gene capture and low-frequency mutation detection.

Description

technical field [0001] The invention relates to the technical field of sequencing, in particular to a micro-DNA targeted enrichment method and its application. Background technique [0002] The target region capture method based on sequencing technology has been widely used in scientific research and clinical gene detection. Since the capture sequencing technology only detects the target region, it improves the utilization rate of sequencing data and reduces the cost of detection. At present, a large number of capture sequencing methods have been developed and widely used in tumor tissue biopsy. With the development of technology, liquid biopsy has received extensive attention from researchers, and some of the research results have been applied to clinical detection and treatment. [0003] Plasma cell-free DNA (cfDNA) carries genetic information and epigenetic information unique to normal cells or tumor cells. Compared with the complexity, hazards, and incompleteness of ti...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2525/191C12Q2525/307C12Q2531/125C12Q2537/1376C12Q2545/101
Inventor 卢文翔王建国白云飞
Owner 徐州海纳生物科技有限公司