DNA (deoxyribonucleic acid) library and preparation method thereof, as well as DNA sequencing method and device

Abstract

The invention belongs to the field of molecular biology and relates to a DNA (deoxyribonucleic acid) library and a preparation method thereof, as well as a DNA sequencing method and device. The preparation method of the DNA library comprises the following steps of: (1) randomly disrupting genomic DNA of a sample into a 20-50kb DNA fragment; (2) performing the following step A or B: (A) filling two tail ends of the disrupted DNA fragment, adding capture marks and then separating the 20-50kb DNA fragment; (B) separating the disrupted 20-50kb DNA fragment, then filling the two tail ends of the DNA fragment and adding the capture marks; (3) performing cyclization on the separated DNA fragment to get annular DNA and removing the non-cyclized DNA fragment; (4) disrupting the annular DNA to a 100-2000bp DNA fragment; and (5) separating the DNA fragment with the capture marks from the DNA fragment obtained in the step (4) so as to get the a captured fragment. The preparation method of the DNA library, disclosed by the invention, has the advantages of being simple, fast and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a DNA library and a preparation method thereof, a DNA sequencing method and a device. Background technique [0002] Next Generation Sequencing (NGS), also known as high-throughput sequencing technology, can simultaneously sequence millions of DNA at a time, which is a revolution in DNA sequencing technology. At present, the three most widely used sequencing platforms are Illumina's GenomeAnalyzer system (that is, Solexa sequencer, which was later developed into HiSeq 2000 system), ABI's SOLiD system, and Roche 454's GS-FLX system. [0003] The high data throughput generated by next-generation sequencing technology makes large-scale genome sequencing possible. However, the sequence read length generated by current high-throughput sequencing technology is much shorter than that of traditional Sanger sequencing (such as ABI 3730xl), less than 200bp, which is unfavorable for genome as...

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Problems solved by technology

[0008] However, the above method is very difficult for constructing a library with inserts up to 20kb or even 50kb, and is more suitable for fragments below 10kb. Moreover, with the help of enzyme cleavage sites or intermediate linkers with biotin labels, the efficiency of enzyme cleavage and linker ligation efficiency, etc. There are uncertain factors and it is difficult to guarantee the success rate

[0009] WO 2010003316A1 discloses a method called parallel sequence tags (GVTs), which uses methylation-sensitive restriction endonucleases to cut different sites to generate different sequence tags to study the methylation of a DNA population. The target DNA population is either randomly fragmented or interrupted at a specific site. The bidirectional GVT generated by the invention is a tag close to the cleavable site of one or more restriction enzymes, and the fragmented target DNA is cloned into a new type of cosmid vector Among them, such as pSLGVT-28, pSLGVT-35, pSLGVT-36, pSLGVT-37 or pSLGVT-38, are used for bidirectional GVT products, using a new generation of SOLEXA, SOLiD or 454 DNA sequencer with a 45-50kb separation length to determine the sequence, but This method will cause the ends of some fosmid clones containing specific regions to be unable to be obtained because the restriction sites of FspB I and Csp6 I used are not evenly distributed in the genome, and there are also problems encountered in the construction of fosmid clones limitations

[0010] Illumina has launched a paired-end library construction kit (Mate Pair Library Kit V2), but this method is only suitable for constructing paired-end libraries with 5-10kb inserts

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