Primer-free gene synthesis method based on plasmid library

A gene synthesis, primer-free technology, applied in the field of genetic engineering, can solve the problems of high error rate and cost limitation, and achieve the effect of low cost, high accuracy and simple operation

Active Publication Date: 2014-07-30
WUHAN GENECREATE BIOLOGICAL ENG CO LTD
View PDF1 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Gene synthesis technology will be applied in more and more fields and play a huge role, but the high error rate and high cost of bases in synthetic genes limit the application of whole g

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
  • Primer-free gene synthesis method based on plasmid library
  • Primer-free gene synthesis method based on plasmid library
  • Primer-free gene synthesis method based on plasmid library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Using the present invention, avidin gene from chicken (G gallus) was synthesized.

[0041] 1. Construct the vector puc-Kana by conventional methods such as PCR, nested PCR, and enzyme-free cloning for use.

[0042] 2. First divide the base sequence (438rnt) of the target gene avidin into 6 segments, each segment is named AV1, AV2, ... AV6, the first 5 segments are 82 nt each, and the sixth segment is 42 nt. To find the corresponding plasmids from the constructed 16384 7bp plasmid library, each 82nt fragment requires 16 plasmids, and the 42nt fragment requires 8 plasmids.

[0043] 3. The 16 plasmids found in each group are divided into two groups, named Ai\B1, A2\B2...A8\B8, and each group of plasmids (An\Bn) has 2bp overlap for ligation , An plasmid and Bn plasmid were reacted according to the following system and procedures.

[0044]

[0045] Both the An group and the Bn group reacted at 37°C for 3h.

[0046] 4. Mix the above digested products with the corresponding...

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 primer-free gene synthesis method based on a plasmid library, and the method is as follows: constructing a plasmid library containing 16384 plasmids (any combination sequence of four basic groups of 47 and 7bp), starting from a constructed plasmid containing 7 bp, dividing a selected target gene according to each 82bp, successively reconstructing four resistance genes Kana, cam, gem and spc by enzyme digestion and enzyme connection operation to obtain a plurality of 82bp plasmids, and assembling the 82bp plasmids into long fragment genes. According to the method, after construction of the plasmid library containing the 16384 plasmids (any combination sequence of four basic groups of 47 and 7bp), various target genes can be synthesized without any primer. The synthesis method has the advantages of simple operation, extremely low mutation rate, no need of sequencing, low cost and less amount of screening, is conductive to constructing a mutant library, can realize solution operation, and is easy to realize automation.

Description

Technical field [0001] The invention relates to a gene synthesis method, which is a primerless gene synthesis method based on a plasmid library, and belongs to the technical field of genetic engineering. Background technique [0002] In recent years, the research progress of synthetic biology is very fast, especially gene synthesis technology. Whole gene synthesis is based on the gene sequence of a certain protein, designing and synthesizing overlapping single-stranded oligonucleotides, and then splicing the full length by overlapping extension PCR method. Gene synthesis does not require a template, and is one of the means to obtain genes. The molecular modification and artificial organization of genes through total gene synthesis is becoming a routine laboratory method. Therefore, it is very important to establish a method that can accurately and efficiently design and synthesize long-segment genes in a relatively inexpensive and short time. [0003] As the research of synthetic...

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
IPC IPC(8): C12N15/10C40B50/06C40B40/02
Inventor 马立新陈晚苹
Owner WUHAN GENECREATE BIOLOGICAL ENG 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