Library construction method for whole genome methylation bisulfite sequencing

A technology for methylation bisulfite and library construction, which can be used in chemical libraries, biochemical equipment and methods, combinatorial chemistry, etc., and can solve problems such as ineffective application

Active Publication Date: 2016-02-24
ZHEJIANG ANNOROAD BIO TECH CO LTD +2
View PDF1 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0019] However, for the initial sample size of the medium level (5 ~ 200ng), neither of the above two methods can be effectively applied

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Library construction method for whole genome methylation bisulfite sequencing
  • Library construction method for whole genome methylation bisulfite sequencing
  • Library construction method for whole genome methylation bisulfite sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Using the method of the present invention to construct a library for whole-genome methylation bisulfite sequencing

[0081] (1) gDNA fragmentation: the initial amount of 100ng genomic DNA was fragmented by an ultrasonic interrupter. Ultrasonic interruption was used to fragment gDNA for 30 seconds, stop for 30 seconds, and repeat 22 times. The interrupted product was purified by 1.8 times Ampure magnetic beads, 20 μL ddH 2 O eluted.

[0082] (2) Bisulfite treatment: EZDNAMethylation-GoldKit (manufacturer Zymo) was used to treat the product in the previous step with Bisulfite and eluted with 31 μLEB.

[0083] (3) One-strand synthesis: react according to the system in the table below. After mixing the first 4 reagents, put them at 65°C for 3 minutes, then drop to 4°C, add 4μL NEBKlenowexo-(28U / μL), and execute procedure 4. Incubate for 5 minutes at ℃, increase the temperature by 0.5 ℃ every 6 seconds until the temperature reaches 37 ℃, and then react at 37 ℃ for 60 minu...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides a library construction method for whole genome methylation bisulfite sequencing. The method is suitable for a medium initial sample size (5-200 ng). The method comprises the steps of obtaining fragmented genome DNAs from 5-200 ng genome DNAs; conducting bisulfite treatment on the fragmented genome DNAs to obtain a single-stranded bisulfite treatment product; synthesizing a first chain by means of an oligo1 primer marked with biotin with the single-stranded bisulfite treatment product as the template; digesting single-stranded DNAs in a system after the first chain is synthesized with exonuclease; obtaining the first chain marked with biotin by means of streptavidin magnetic beads; synthesizing a second chain by means of an oligo2 primer with the first chain marked with biotin as the template; conducting PCR amplification with the second chain as the template, so that a DNA library for next-generation sequencing is constructed.

Description

Technical field [0001] The invention belongs to the field of gene detection, and specifically relates to a method for constructing a library for whole genome methylation bisulfite sequencing suitable for an initial sample amount of a medium level (5 to 200 ng). Background technique [0002] DNA methylation is one of the most common epigenetic modifications of genomic DNA. A large number of studies have shown that DNA methylation modifications are essential for maintaining normal cell functions, transmitting genomic genetic imprints, embryonic development, and human tumorigenesis. effect. With the birth of high-throughput sequencing, the research on DNA methylation has become more and more in-depth. As the gold standard for DNA methylation detection, whole-genome bisulfite sequencing (WGBS or BS-Seq, WholeGenomeBisulfateSequencing) [1] , Can analyze the whole genome DNA methylation, and has the single-base level detection sensitivity. However, the initial volume of BS-Seq technol...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68C40B50/06
Inventor 洪燕徐护朝王晓雯赵红梅张广思玄兆伶李大为梁峻彬陈重建
Owner ZHEJIANG ANNOROAD BIO TECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap