Preparation method of double-stranded DNA probe for liquid-phase hybrid capture and sequencing

Abstract

The invention discloses a preparation method of a double-stranded DNA probe for liquid-phase hybrid capture and sequencing. The method comprises the following steps of: designing and synthesizing target region specific primers and 5'-biotin modified universal primers, amplifying original probes through a first round of PCR by taking the specific primers and DNAs containing a targeting sequence astemplates; then, amplifying biotin-modified probes by using the universal primers and the original probes as templates; purifying each probe with qualified quality control by using nucleic acid purification magnetic beads; performing balanced mixing on each probe to form a probe pool; and subjecting the probe pool to heating denaturation so as to finish the preparation process of the double-stranded DNA probe. According to the method, the double-stranded DNA probe with a length of 240 bp can be prepared without depending on an expensive synthesis platform; probe synthesis efficiency is not influenced by GC content and length; preparation process is simple; and preparation cost is low. Each probe is independently prepared and subjected to quality control, so the abundance of each probe in the probe pool is consistent. When the double-stranded DNA probe is used, harsh environment and operation requirements of RNA probes are discarded, and positive and negative double strands of DNAs of atarget library can be captured at the same time.

Description

technical field [0001] The invention belongs to the technical field of high-throughput gene sequencing, and in particular relates to a method for preparing a double-stranded DNA probe for liquid-phase hybridization capture sequencing. Background technique [0002] In 2005, life sciences launched the 454 FLX pyrosequencing platform, which opened a new era of high-throughput gene sequencing. The most notable feature of high-throughput sequencing technology is the simultaneous sequencing of millions or more DNA molecules in parallel, which is a revolutionary change to the traditional Sanger sequencing technology. The completion of the Human Genome Project based on Sanger sequencing technology took 10 years and cost tens of billions of dollars. However, Illumina's high-throughput sequencing platform has achieved a 24-hour cost of about 10,000 yuan to complete a person's whole genome resequencing. In the field of gene function research and application, high-throughput sequencin...

AI Technical Summary

Problems solved by technology

At present, the probe preparation methods based on the above two oligonucleotide synthesis techniques all have the following problems: the error rate of artificial synthesis is high; the efficiency of synthesis is affected by the GC content and sequence length of the sequence; , the lower the synthesis efficiency; the maximum length of synthesis is 120nt; the abundance gap of each probe in the probe pool is large, and multiple PCR amplifications will further increase the gap in probe abundance, affecting the balance of captured sequencing data ; The prepared RNA probe has poor stability and has high requirements for the use environment

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