Preparation method of nucleic acid targeted capture sequencing library based on long chain molecule inversion probe

Abstract

The invention discloses a preparation method of a nucleic acid targeted capture sequencing library based on a long chain molecule inversion probe. The preparation method comprises the following steps:a, synthesizing a capture probe A, a capture probe B and a connexon C; b, adding phosphorylated probes A and B and the connexon C into a ligase reaction system, simultaneously adding DNA (Deoxyribonucleic Acid) ligase so as to connect the A with the B under the bridging effect of the C; c, combining multiple connection mixtures for different target areas, and separating and purifying the connected product by denaturing electrophoresis or a nucleic acid purification kit to obtain the long chain molecular inversion probe; d, mixing the long chain molecular inversion probe with DNA or cDNA of ato-be-tested sample, hybridizing, adding DNA polymerase, DNA ligase, dNTP and a Mg2<+>-containing buffer solution into a buffer solution, extending the long chain molecular inversion probe and formingclosed molecules under the action of the DNA ligase; e, adding exonuclease to degrade non-cyclic DNA molecules; f) carrying out PCR (Polymerase Chain Reaction) amplification by using primers corresponding to a common sequence region of the long-chain molecular inversion probe to obtain the sequencing library of a 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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