A method for constructing a large fragment library and its application

Abstract

The invention relates to a method for constructing a large-segment DNA library and its application. The method for constructing a large-fragment DNA library provided by the invention is used for the construction of a large-fragment DNA library. The method for constructing a large fragment DNA library comprises the steps of adding a large blunt-ended DNA fragment to a phosphorylated adapter at the 5' end to obtain a large DNA fragment with an adapter, and circularizing the large DNA fragment with an adapter to obtain a large circularized DNA fragment and A step of a mixture of large non-circularized DNA fragments, and a step of subjecting the mixture to linear DNA digestion to obtain large circularized DNA fragments, wherein the linear DNA digestion uses an exonucleic acid having a specific digestion function of phosphorylated DNA at the 5' end Dicer.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a method for constructing a large fragment DNA library, the large fragment DNA library constructed by the method and the application of the large fragment DNA library in sequencing. Background technique [0002] For a species whose genome sequence is unknown or has no genome information of related species, its genome DNA fragments of different lengths and their libraries are sequenced, and then spliced, assembled and annotated by bioinformatics methods to obtain the complete genome of the species Sequence map, called genome de novo sequencing, also called de novo sequencing. Today, with the rapid development of genomics, the combination of de novo sequencing and comparative genomic methods can be used to explore the origin and evolution of the species, and to study the molecular mechanisms of its growth and development, shape production and environmental adaptation, w...

AI Technical Summary

Problems solved by technology

The disadvantage of next-generation sequencing is the short read length. The Roche 454, which has the longest read length in the second-generation sequencer, can only perform DNA sequencing of up to 400 bp. According to the overlapping sequences on different small fragments, their sequencing structures are spliced ​​into contigs of different sizes (fragment contigs)

This digestion method cannot specifically digest uncircularized DNA fragments containing biotinylation, and is a non-specific linear digestion method

Moreover, the digestion method using plasma safe ATP-Dependent DNase needs to add ATP as the reaction kinetic energy and exonuclease I as an auxiliary to be implemented, which will bring higher costs for manpower, reagents and reagent storage

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