Sequencing method based on gene capture technology

Abstract

The invention provides a sequencing method based on a gene capture technology. The sequencing method has the principle that a recombinase compound is used for assisting a single-stranded DNA probe with a closed 3' end to efficiently and specifically hybridize into a double-stranded target DNA molecule under the condition of 37 DEG C, and then an affinity label on the single-stranded DNA probe is used for carrying out specific enrichment on the target DNA. The method not only has the advantages of relatively strong long probe pairing, low probe design requirement and the like of a probe hybridization capture technology, but also has the advantages of simplicity in operation, short time consumption, no need of thermal denaturation of DNA, high capture efficiency, low cost and the like of a CRISPR / dCas9 system, and the probe does not participate in subsequent amplification, so that the identification accuracy is improved. Therefore, compared with other capture technologies, the RATE-seq has the obvious advantages of simplicity in operation, high capture efficiency, extremely short consumed time (30 minutes), good stability, low cost and the like, and is very suitable for gene capture and automatic application. In addition, the RATE-seq can effectively separate and remove human host DNA in a pathological sample, and the detection rate of pathogenic microorganisms is increased.

Description

technical field [0001] The patent of the present invention relates to a sequencing method based on gene capture technology, which belongs to the field of biotechnology. Background technique [0002] The second-generation and third-generation sequencing technologies have become the core technologies in the field of gene diagnosis and treatment due to their advantages of large throughput, complete information, and easy operation. Gene trapping has been a major challenge for DNA and RNA high-throughput sequencing technologies. Due to the huge amount of human genome information, which contains about 3 billion base pairs, the real coding gene region accounts for less than 4%, and the exon region and transcription regulatory element region account for less than 1%. The vast majority of pathogenic mutations are enriched in exon regions and transcriptional regulatory element regions, so the genetic information in these regions plays a key role in the diagnosis and treatment of dise...

AI Technical Summary

Problems solved by technology

Compared with the traditional probe hybridization capture technology, this capture method has the advantages of low cost, short time-consuming (2h), high capture efficiency, simple operation, and no need for heat denaturation of DNA, but the non-specific binding of dCas9 itself to DNA and Off-target effects of sgRNAs severely hamper capture efficiency and specificity

In addition, the design of sgRNA is difficult (depending on the PAM sequence), and the pairing length with the target site is short (20 nt), which seriously hinders its application in large-scale gene capture.

Moreover, the poor stability of sgRNA and dCas9 also makes it difficult for this capture method to be widely promoted and popularized.

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