Construction method of single stranded DNA next generation sequencing library specific to cfDNA

A technology for a second-generation sequencing library and a construction method, which is applied in the field of construction of a single-stranded DNA second-generation sequencing library for cfDNA, can solve the problems of complex steps, long connection time, low connection efficiency, etc., and achieves short library construction time and enrichment. The effect of high efficiency and low cost of database construction

Active Publication Date: 2017-07-28
SHENZHEN HAPLOX BIOTECH
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Problems solved by technology

The ligation efficiency of this enzyme is relatively low, requires a long time for ligation, and requires biotin-labeled magnetic beads. The cost

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  • Construction method of single stranded DNA next generation sequencing library specific to cfDNA

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Embodiment Construction

[0035] The present invention will be described in further detail below through specific examples. Unless otherwise specified, the instruments, equipment and reagents used in the following examples are all available to those skilled in the art through public channels such as commercial purchase.

[0036] Explanation of terms:

[0037] bufeer is buffer; to is added to the total content; SAP is Super Absorbent Polymer, super absorbent resin; the size of x is 0

[0038] see figure 1 As shown, a method for constructing a single-stranded DNA next-generation sequencing library for cfDNA comprises the following steps:

[0039] 1. cfDNA dephosphorylation treatment. The reaction system is as follows:

[0040] SAP buffer 2μl

[0041] cfDNA x μl

[0042] wxya 2 O to 20μl

[0043]React at 37°C for 30-60min for dephosphorylation reaction, then react at 65°C for 15-30m...

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Abstract

The invention discloses a construction method of single stranded DNA next generation sequencing library specific to cfDNA. The construction method includes steps of 1), cfDNA extraction; 2), dephosphorylation; 3), denaturation to be single stranded cfDNA; 4), intermolecular single stranded connection on 3' end single stranded DNA interface; 5), extension: the acquired single stranded connection product is a template, and the known sequence design primer of the 3' end interface is extended; 6), intermolecular double stranded connection between the acquired double stranded DNA product and a 5' end double stranded DNA interface; 7), PCR amplification: use the acquired complete double stranded DNA interface connection product as a template, and take the known sequences of the interfaces at 3' end and the 5' end as forward and reverse products, and perform PCR amplification; 8), PCR product purification by a paramagnetic particle method, and library recycling. The original cfDNA gathering efficiency is high, and the double stranded, the single stranded and damaged DNA can be effectively detected.

Description

technical field [0001] The invention relates to the field of construction of a second-generation gene sequencing library, in particular to a method for constructing a single-stranded DNA next-generation sequencing library for cfDNA. Background technique [0002] cfDNA (cell-free circulating DNA) generally refers to DNA fragments present in plasma, urine and other body fluids, composed of double-stranded DNA, single-stranded DNA and damaged DNA, which are used in tumor monitoring, non-invasive prenatal and organ transplantation, etc. There are a wide range of applications in the field. [0003] Most of the current cfDNA next-generation sequencing technologies use conventional double-stranded DNA library construction methods, which can only detect undamaged double-stranded DNA, and in order to remove adapter dimer contamination, it is generally necessary to use magnetic beads The step of fragment selection, which leads to the loss of information of some small fragments of dou...

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C40B50/06
CPCC12N15/1093C12Q1/6806C12Q1/6869C40B50/06C12Q2521/525C12Q2525/191C12Q2521/501C12Q2531/113C12Q2535/122
Inventor 温媛刘明王一杏毛燕
Owner SHENZHEN HAPLOX BIOTECH
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