Primer-free gene synthesis method

A gene synthesis and gene technology, applied in the field of library-based primer-free gene synthesis, can solve the problems of high synthesis cost, high error rate, and reduced error rate, and achieve the effects of high splicing accuracy, low synthesis cost, and cost reduction

Inactive Publication Date: 2014-10-22
WUHAN GENECREATE BIOLOGICAL ENG CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This solid-phase synthesis technology greatly reduces the error rate of synthesis, but due to the need to use specially modified primers in the synthesis process, this will lead to an increase in the cost of primers, which will lead to an increase in the cost of subsequent gene synthesis
At the same time, solid-phase synthesis technology relies on special instruments and equipment, which cannot meet the needs of ordinary laboratories and scien

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 constructs pNew carrier plasmid

[0049] 1. Construct the linear vector backbone pNew

[0050] Using the commercialized vector pet23a plasmid as a template and ori-PF / ori-PR as primers (see Table 1 for the sequence), the linear vector backbone pNew (1.9kb) was obtained by PCR amplification (its nucleotide sequence is SEQ ID NO. 21), the recognition site of restriction endonuclease BseRI was introduced by primer ori-PR in the PCR process.

[0051] 2. Construction of large fragment 5SGCK

[0052] Design primer 5Spc-PF / 5Spc-PR (sequence is shown in Table 1), take RNALIC plasmid as template, obtain 5Spc fragment (656bp) (its nucleotide sequence is shown in SEQ ID NO.22) by PCR amplification, amplify The 5' and 3' ends of the good 5Spc fragments were respectively equipped with restriction enzyme sites BsgI and XbaI; design primers 5Gem-PF / 5Gem-PR (see Table 1 for the sequence), and use the pDEST32 plasmid as a template to amplify by PCR The 5Gem fragment (535...

Embodiment 2

[0060] Synthesis of embodiment 2 target gene

[0061] 1. Construction of plasmid library

[0062] The pNew vector constructed in Example 1 was constructed by conventional construction methods of PCR, reverse amplification vector, and enzyme-free cloning 4 7 (16384) kinds of plasmid libraries, each plasmid in the plasmid library randomly contains any permutation and combination sequence of 7 base pairs.

[0063] 2. Analysis and grouping of target genes

[0064] Select the target gene X to be synthesized, divide the pre-synthesized target gene DNA sequence according to 82bp of each fragment, and name each fragment G1, G2, G3, G4, G5..., between each fragment There are 3 base repeats.

[0065] 3. Library plasmid search

[0066] According to the one-to-one correspondence between the sequences of small fragments such as G1 and G2 and the corresponding numbers of the plasmids, find the corresponding plasmids in the established 7bp plasmid library, and each 82bp fragment requires...

experiment example 1

[0074] The gene synthesis of experimental example 1 avidin (avidin)

[0075] A gene fragment of avidin derived from chicken (G. gallus) was synthesized by using the gene synthesis method of the present invention.

[0076] The synthesis process of avidin includes the following steps:

[0077] (1) The commercially available puc-19 plasmid and pGSilG-Lic plasmid (purchased from Invitrogen) were used to construct the puc-Kana plasmid vector by conventional methods such as PCR, nested PCR, and enzyme-free cloning.

[0078] (2) Divide the base sequence (438bp) of the target gene avidin into 6 segments, and each segment is named AV1, AV2, ... AV6, each of the first 5 segments is 82bp, and the sixth segment is 42bp. Then, find out the corresponding plasmids in the constructed 16384 7bp plasmid libraries, each 82bp fragment needs 16 plasmids, and each 42bp fragment needs 8 plasmids.

[0079] (3) The 16 plasmids found in each group are grouped in pairs and named A1\B1, A2\B2...A8\B8. ...

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Abstract

The invention discloses a primer-free gene synthesis method and belongs to the field of artificial synthesis of genes. The invention discloses a pNew carrier plasmid, and the pNew carrier plasmid has a nucleotide sequence shown in the formula of SEQ ID NO.33. The invention also discloses a carrier bank which is constructed from pNew carrier plasmids and contains 16384(47) plasmids. The invention further discloses a primer-free gene synthesis method. The primer-free gene synthesis method comprises the following steps of 1, carrying out grouping on targeted gene DNA sequences to be synthesized according to a ratio of 82bp per fragment, 2, finding plasmids corresponding to the grouped fragments in the constructed plasmid bank, 3, carrying out enzyme digestion on the corresponding plasmids, carrying out screening by antibiotic and carrying out reconstruction to obtain a plasmid containing 82bp of the targeted gene sequences, and 4, splicing the gene fragments by a golden-gate cloning reaction to obtain a complete targeted gene. The primer-free gene synthesis method is free of primers, has a low synthesis cost, a very low mutation rate and high accuracy, can synthesize special gene sequences such as a highly repetitive sequence and Poly A, has simple processes and can realize automatic operation.

Description

technical field [0001] The invention relates to a gene synthesis method, in particular to a library-based primer-free gene synthesis method, which belongs to the technical field of gene synthesis. Background technique [0002] In recent years, research on synthetic biology, especially gene synthesis technology, has progressed very rapidly. At present, the whole gene synthesis is based on the nucleotide sequence of a certain protein, firstly designing and synthesizing overlapping single-stranded oligonucleotides, and then splicing the full-length gene sequence by overlapping extension PCR method. Template is currently one of the effective means to obtain genes. [0003] So far, the gene synthesis methods that have been reported all rely on chemically synthesized oligonucleotide primers, mainly including the following methods: (1) chemical synthesis, because the principle of chemical synthesis itself has defects to a certain extent, These defects determine that the DNA fragm...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/10C40B40/06
CPCC12N15/10C12N15/63C40B40/02C40B50/06
Inventor 马立新沈鹤霄陈晚苹华权高张桂敏杨明波陈羽西
Owner WUHAN GENECREATE BIOLOGICAL ENG CO LTD
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