Establishment method of long mate pair library

A construction method and long-fragment technology, applied in the effective application field of de novo sequencing and assembly, can solve the problems of complex experimental process, difficult control, high error rate, etc., and achieve the effect of good reproducibility, easy operation, and simple steps

Inactive Publication Date: 2013-01-09
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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Problems solved by technology

At present, several (long mate pair library construction methods have disadvantages such as low efficiency, complicated experimental process, difficult control, high error rate, chimeric molecules, and low library ...

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  • Establishment method of long mate pair library

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Embodiment 1

[0016] Example 1 Construction of LMP library

[0017] 1) First, the genomic DNA is randomly interrupted, and the interrupted DNA is repaired by end gap filling;

[0018] First, the integrity of the extracted genomic DNA was detected by 1.2% agarose gel electrophoresis, and Qubit (Invitrogen) was used for quantitative and qualitative analysis to ensure the high quality of the DNA, including integrity and purity. Then, take 120 μl of genomic DNA (20-35 ng / μl), and use CovarisS220 to break up the fragments. The specific conditions are as follows: 7°C, mini blue tube, Duty Factor 20%, Peak Incident Power 3W, cycles per Burst 1000, 900 Second. After ultrasonic crushing, use 1.2% agarose gel electrophoresis to recover the 2 Kb target region, and use T4 DNA polymerase to repair the recovered fragments. The 200 μl system contains the following components: 100 μl recovered 2 Kb DNA fragments , 10 mM dNTP 8 μl, T4 DNA polymerase (3 U / μl) 5 μl, PNK (poly nucleotide kinase) (10 U / μl) 1....

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Abstract

The invention relates to an establishment method of a long mate pair library. The establishment method comprises the steps as follows: 1) randomly interrupting DNA (deoxyribose nucleic acid), and completing and repairing the gaps at the tail end; 2) preparing A-Fragment; 3) carrying out cohesive end connection to A-Fragment and LMP Adaptor; 4) digesting the LMP-Adaptor-Fragment with at least two restriction enzymes which can distinguish four basic groups; 5) cyclizing the fragments in the obtained enzyme digesting library again; and 6) amplifying the purified ring molecule through primer of the LMP Adaptor; and recovering the amplified products, thus finishing the establishment of LMP (long mate pair) library. According to the library establishment method provided by the invention, only two 63bp oligonucleotides sequences are needed to be synthetized, and simple experiments such as molecular biology connection and amplification are carried out, and the two ends of the obtained LMP library have the sequencing primer sequences which can be directly applied to next generation sequencing; and moreover, the fragment subjected to secondary cyclizing has the known restriction enzyme site, and the sequences of two ends of the to-be-tested fragments can be quickly sorted, the sequence testing data can be effectively sieved, and the mosaic sequence can be removed.

Description

technical field [0001] The invention belongs to the technical field of molecular biology sequencing, and in particular relates to a method for constructing a long fragment terminal library (Long Mate Pair, LMP), and the effective application of the method in genome sequence splicing, especially in de novo sequencing and assembly. Background technique [0002] At present, the next-generation sequencing method has been widely used in genome de novo assembly. Due to the huge sequencing data and short sequencing length, the key factor affecting the quality of assembly is the repetitive sequence of the genome (such as microsatellite sequence, ribosomal RNA sequence, transposition subsequence, etc.), the length of the repeating unit ranges from several bp to several kb. Constructing a library of insert fragments of different lengths for sequencing can span repeat sequences of different lengths and effectively improve the integrity and accuracy of genome assembly. LMP is based on ...

Claims

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Application Information

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IPC IPC(8): C40B40/06C40B50/06C12Q1/68
Inventor 陈祖耕姜楠王萍暴云娟刘桂友陈晓云
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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