Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Construction method of genome-wide mRNA 3' end gene library

A technology of gene library and whole genome, applied in the field of preparation of whole genome mRNA 3' end library, can solve the problems affecting the sequencing process and results, and achieve the effect of increasing anchoring efficiency, simple sample preparation steps, and ensuring integrity.

Inactive Publication Date: 2014-10-22
SUN YAT SEN UNIV
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutations in the 3'UTR can affect the expression of one or more genes, leading to disease
Similar to the 454 GS FLX, if you encounter a few or even a dozen consecutive bases, the fluorescent signal at the same position is too strong, which will also affect the sequencing process and results

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
  • Construction method of genome-wide mRNA 3' end gene library
  • Construction method of genome-wide mRNA 3' end gene library
  • Construction method of genome-wide mRNA 3' end gene library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 as figure 1 As shown, in another embodiment of the present invention, the flow chart of the construction of the mRNA 3' end library, the specific process is: (1) take 10ug / 10ul of DNase-digested total RNA, and fragment it to the expected size by high temperature (2) using template switching technology (Template Switching Technology), the Oligo d(T) primer containing the Solexa or 454 sequencing adapter sequence is anchored to the Poly of mRNA in the total RNA obtained in step (1) by reverse transcription (A) Tail, reverse transcriptase Super ScriptII adds 3-6 cytosines (CCC) to the end of a synthetic cDNA strand; (3) Adds Solexa with 3 ribonucleic acid guanines (GrGrGr) at one end Or 454 sequencing primers, the guanine sequence (GrGrGr) is anchored to the cytosine sequence (CCC) of one strand of cDNA, and then under the action of reverse transcriptase, the other sequence of (CCC) on the first strand of cDNA is formed by the principle of base complementarity S...

Embodiment 3

[0049] Example 3 Establishment of a 454 sequencing library at the mRNA 3' end of the human breast cancer cell line MCF7.

[0050] 1) Reagents and instruments PCR instrument was used for high-temperature fragmentation of RNA; RNase inhibitor was purchased from Toyobo; reverse transcriptase SuperScriptII TM Sorbitol, D-(+)-trehalose was purchased from Sigma; T4Gene 32 protein was purchased from NEB; PCR reaction Taq high-fidelity DNA polymerase was purchased from Invitrogen. Plasmid Plate Extraction Kit was purchased from Axygen; MinElute PCR Purification Kit, MinElute PCR Purification Kit was purchased from Qiagen; Bioanalyzer is purchased from Agilent; Fluorometer and each primer were purchased from Invitrogen.

[0051] 2) Experimental method (1) Random fragmentation of total RNA For total RNA digested with DNase genomic DNA, 10ug / 10ul was taken and interrupted at 94°C for 30-40 minutes to generate uniform random fragmented total RNA. This method can avoid the use of hi...

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 relates to a preparation method of a terminal gene library of full genome mRNA3' and is suitable for high-throughput sequencing. A cDNA-chain is synthesized by transforming a template, and mutation is induced into polymerase chain reaction (PCR) to obtain a non-translated regional deoxyribonucleic acid (DNA) sequence of the full genome mRNA3'. The data yield of the library prepared by the preparation method of the terminal library of the full genome mRNA3' is large; the sample preparation steps are simple and economic; and the preparation method is beneficial for analysis of later-stage bioinformatics, and is easy for large-scale applications.

Description

technical field [0001] The invention relates to a method for constructing a gene library, in particular to a method for preparing a whole-genome mRNA 3' end library suitable for high-throughput sequencing. Background technique [0002] The 3' untranslated region (3'UTR for short) is a special part of messenger RNA (mRNA) located at the 3' end of the coding region of a gene. 3'UTR is an important regulatory element of mRNA function, and has the following regulatory sequences: polyadenylation site, usually AAUAAA; protein binding site and miRNA target site, etc. 3'UTR can not only control the in vivo stability and degradation rate of mRNA, regulate the subcellular localization and translation level of mRNA, but also determine the type of cell it is expressed in, control the utilization efficiency of mRNA, and assist in the identification of special codons. Mutations in the 3'UTR can affect the expression of one or more genes, leading to disease. Human genes often have multip...

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 Patents(China)
IPC IPC(8): C12Q1/68C40B50/06C40B40/06
Inventor 徐安龙付永贵孙雨李宇新万谅饶兴蔷
Owner SUN YAT SEN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com