Nucleic acid target capture sequencing library preparation method based on long-chain molecular inversion probe

Abstract

The invention discloses a method for preparing a nucleic acid targeted capture sequencing library based on long-chain molecular inversion probes; a) synthesizing capture probe A, capture probe B and linker C; b) adding phosphoric acid to the ligase reaction system Ligated probes A, B and linker C, while adding DNA ligase to connect A and B under the bridging action of C; c) Merge multiple ligation mixtures for different target regions, and use denaturing electrophoresis or nucleic acid purification reagents Cartridge separation and purification of the connected product to obtain long-chain molecular inversion probes; d) long-chain molecular inversion probes are mixed with the DNA or cDNA of the sample to be tested, hybridized, and DNA polymerase, DNA ligase, dNTP and Mg2+-containing buffer extend the long-chain molecule inversion probe, and form a closed molecule under the action of DNA ligase; e) add exonuclease to degrade the DNA molecule without circularization; f) use and long-chain molecule inversion The primers corresponding to the common sequence region of the probe were amplified by PCR to obtain a sequencing library of the targeted region.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid determination or inspection methods, and in particular relates to a method for preparing a nucleic acid target sequence capture sequencing library based on primer extension. Background technique [0002] The new generation of high-throughput DNA sequencing technology that has emerged in recent years can sequence and quantify billions of DNA fragments in parallel, providing a powerful tool for basic biomedical research and clinical testing; high-throughput DNA sequencing technology The development has also led to the rise of another important technology-target sequence capture sequencing; target sequence capture sequencing is to first extract the DNA fragments of the target genes we care about through some targeted methods to prepare a target sequence sequencing library, and then through Qualcomm Quantitative sequencing to analyze it, such as exome (Exome) capture sequencing to capture and det...

AI Technical Summary

Problems solved by technology

But its main disadvantage is that the region captured by the probe is limited, and the capture region is generally 40-170bp; if it exceeds 170bp, the capture efficiency is very low

At present, the read length of high-throughput sequencing can reach 2×250bp; in addition, traditional MIP or Padlock probes cannot accurately quantify the captured DNA fragments, and the limited capture length and inaccurate quantification will limit MIP or Padlock probes Applications

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