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US 11,001,837 B2Granted
Low-frequency mutations enrichment sequencing method for free target DNA in plasma
AssigneeGeneplus - Beijing
InventorsXiaoxing Lv, Xin Yi, Meiru Zhao, Yanfang Guan, Tao Liu, Ling Yang
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Abstract
The present invention provides a low-frequency mutation enrichment sequencing method for free target DNA in plasma, comprising plasma DNA extraction and library construction, general library TT COLD PCR amplification enrichment, probe enrichment capture, PCR and sequencing of captured products, and positive and negatice double-strand error-correction low-frequency information analysis.
Claims9
A method for identifying low-frequency mutations in a target DNA present in plasma obtained from human peripheral blood, comprising the following steps:
- (1) extracting the target DNA from the plasma and constructing a DNA library containing fragments from the extracted target DNA;
- (2) amplifying and enriching such DNA fragments in the library containing predetermined mutations by first determining the Tm value of the library followed by universal library TT-COLD PCR;
- (3) enriching for fragments containing low-frequency mutations using hybridization capture probes and then amplifying and sequencing the resulting hybridization capture products which include fragments containing low-frequency mutations;
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- (4) analyzing the sequences of the fragments containing the low-frequency mutations using a forward and reverse double-strand error correction procedure so as to identify low-frequency mutations in the target DNA.
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