Solid-phase-carrier-membrane-based target nucleic acid enrichment method for high-throughput sequencing

Abstract

The invention relates to the field of biotechnology, and specifically provides a solid-phase-carrier-membrane-based target nucleic acid enrichment method for high-throughput sequencing. The solid-phase-carrier-membrane-based target nucleic acid enrichment method comprises: 1) combining a nucleic acid probe library and a solid phase carrier membrane to form a probe-solid phase carrier membrane complex; 2) adding target nucleic acid to a hybridization solution containing the probe-solid phase carrier membrane complex, and enriching the target nucleic acid; 3) washing the obtained probe-solid phase carrier membrane complex with the target nucleic acid by using a washing solution, eluting from the solid phase carrier membrane by using an eluent, and purifying the enriched target nucleic acid library; and 4) applying the target nucleic acid library enriched in the step 3) in high-throughput sequencing. According to the present invention, with the method, the high-multiple coverage and single-base displacement nucleic acid probe for enriching the target nucleic acid can be rapidly produced, and is immovably fixed on the solid phase carrier membrane; and the method has advantages of simple operation, flexible probe acquisition and low cost.

Description

technical field [0001] The present invention relates to the field of biotechnology, and in particular provides a method for enriching target nucleic acid based on a solid-phase carrier membrane for high-throughput sequencing. Background technique [0002] With the development of high-throughput sequencing, genome sequencing has become the most important method for high-throughput and high-depth identification of related loci variants, thereby identifying potential diseases, and has a wide range of applications in scientific research. In medical diagnosis, high-throughput sequencing can directly obtain a large amount of relevant locus variation information, which is of great significance for precision medicine. However, based on the current situation, most research targets and diagnostic targets only account for a small part of the overall sample. For example, the vast majority of known disease-related loci variants exist in a specific region of the genome, such as the exome,...

AI Technical Summary

Problems solved by technology

In this method, the hybridization time and conditions of the target nucleic acid and the probe nucleic acid cannot be well regulated, resulting in high background noise

[0013] CN101351564A discloses a nucleic acid detection by target-specific hybridization method. Although the method uses a solid phase carrier to enrich target nucleic acid: probe nucleic acid hybridization complex, and then obtain target nucleic acid, the generated target nucleic acid: probe The nucleic acid complex is an RNA:DNA complex, and the target nucleic acid is RNA instead of DNA, which greatly narrows the applicable range

Secondly, this method needs to carry out certain special modification to solid-phase carrier, and then enriches target nucleic acid: probe nucleic acid complex, has increased cost

At the same time, this method is only for the capture of specific sites, and is not suitable for the requirements of high-throughput large-scale enrichment

[0014] At present, solid-phase carrier membranes such as nylon membranes, cellulose acetate membranes, or the like are used as media to enrich target nucleic acid libraries and finally use them in high-throughput sequencing services. There are no reports and studies on this aspect.

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