Method of establishing DNA large fragment library and applications thereof

Abstract

The invention relates to a method of establishing a DNA large fragment library and applications thereof. The provided method is used to establish a DNA large fragment library. According to the method,a 5'-terminal phosphorylation joint is connected to the blunt end of a DNA large fragment to obtain a DNA large fragment with a joint; the DNA large fragment with a joint is subjected to cyclizationto obtain a mixture of cyclized DNA large fragments and non-cyclized DNA large fragments; the mixture is subjected to linear DNA digestion to obtain cyclized DNA large fragments, and in linear DNA digestion, exonuclease with a function of specifically digesting phosphorylated DNA in the 5'-terminal is adopted.

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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