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Improved overlap extension PCR process and mutation gene obtained thereby

A technology of overlapping extension and gene mutation, applied in fermentation and other directions, can solve the problems of cumbersome test steps and heavy workload, and achieve the effects of increasing accumulation, good fidelity and high-efficiency expression

Inactive Publication Date: 2006-11-15
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although OE-PCR has been widely used as a common method, in general, only one mutation can be introduced at a time, and when multiple sites need to be mutated, cumbersome experimental steps and a large workload will be required , which also increases the possibility of introducing some unnecessary point mutations

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  • Improved overlap extension PCR process and mutation gene obtained thereby
  • Improved overlap extension PCR process and mutation gene obtained thereby
  • Improved overlap extension PCR process and mutation gene obtained thereby

Examples

Experimental program
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Effect test

Embodiment 1

[0032] A gene sam1 encoding SAM synthetase (2.5.1.6) derived from Saccharomyces cerevisiae INVScI strain has the base sequence in SEQ ID No: 1 in the sequence table.

[0033] SEQ ID No: 1:

[0034] ATGGCCGGTACATTTTTATTCACTTCTGAATCCGTTGGTGAAGGTCACCCGAGA

[0035] TAAGATCTGTGACCAAGTTTCCGACGCCATCTTGGACGCTTGTTTAGCCGAGG

[0036] ACCCTCACTCCAAAGTTGCGTGTGAAACCGCGGCAAAGACTGGTATGATTATG

[0037] GTCTTTGGTGAAATTACTACCAAGGCACAGTTGGATTACCAAAAAATCGTCAG

[0038] AGACACCATCAAGAAGATTGGTTACGATGATTCCGCCAAGGGTTTCGACTATA

[0039] AGACCTGTAACGTCCTTGTCGCCATTGAGCAACAATCTCCAGATATCGCCCAA

[0040] GGTGTCCACGAGGAGAAGGATTTGGAAGACATCGGTGCCGGTGACCAAGGTAT

[0041] CATGTTTGGTTACGCCACAGATGAAACTCCAGAGGGTTTGCCTTTGACTATTC

[0042] TTTTGGCTCATAAACTAAACATGGCCATGGCTGACGCGAGAAGAGATGGCTCT

[0043] TTAGCGTGGTTGAGACCAGACACCAAGACTCAAGTCACCGTCGAATACAAGGA

[0044] TGACCACGGTAGATGGGTTCCACAAAAGAATCGACACCGTCGTCGTCTCCGCTC

[0045] AACATGCTGACGAAATCACGACCGAGGACTTAAGAGCGCAACTAAAGTCCGAG

[0046] ATCATTGAAAAAGTCATCCCAAGAG...

Embodiment 2

[0072] The sam1 is shuffled by using a new comprehensive gene shuffling method to obtain the gene sam1 encoding a recombinase with improved enzyme activity, which has the base sequence of SEQ ID No: 3 in the sequence table.

[0073] ATGGCCGGTACATTTTTATTCACTTCTGAATCCGTTGGTGAAGGTCACCCGAGA

[0074] TAAGCTCTGTGACCAAGTTTCCGACGCCATCTTGGACGCTTGTTTAGCCGAGG

[0075] ACCCTCACTCCAAAGTTGCTTGTGAAACCGCTGCAAAGACTGGTATGATTATG

[0076] GTCTTTGGTGAAATTACTACCAAGGCACAGTTGGATTACCAAAAAATCGTCAG

[0077] AGACACCATCAAGAAGATTGGTTACGATGATTCCGCCAAGGGATTCGACTATA

[0078] AGACCTGTAACGTCCTTGTCGCCATTGAGCAACAATCTCCAGACATCGCCCAA

[0079] GGTGTCCACGAGGAGAAGGATTTGGAAGACATCGGTGCCAGTGACCAAGGTAT

[0080] CATGTTTGGTTACGCCACAGATGAAACTCCAGAGGGTTTGCCTTTAACTATTC

[0081] TTTTGGCTCATAAACTAAACATGGCCATGGCTGACGCTAGAAGAGATGGCTCT

[0082] TTAGCTTGGTTGAGACCAGACACCAAGACTCAAGTCACCGTCGAATACAAGGA

[0083] TGACCACGGTAGATGGGTTCCGCAAAGAATCGACACCGTCGTCGTCTCCGCTC

[0084] AACATGCTGACGAAATCACTACCGAGGACTTAAGAGCTCAACTAAAAGTCCGAG

...

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Abstract

The invention relates to the method of gene fixed-point mutation which is a modified overlap extension PCR method and the mutated gene. The process includes the fragment composition, the double mixing, the prepared extension, the whole DNA composition and after extension. The method is detected by the synzyme gene sam1 from the SAM of the Saccharomyces cerevisiae and gets the mutated gene modified by the rare coden. The SAM synzyme encoding by the gene can express in the yeast and improves the yield of the intracellular SAM. The invention can reach the many fixed-point mutations in a same reaction process.

Description

technical field [0001] The invention relates to a gene site-directed mutation method, specifically an improved overlap extension PCR method and the obtained mutant gene, and a gene modification method capable of realizing multi-point site-directed mutation in one reaction process. Background technique [0002] Site-directed mutagenesis (SDM) refers to the introduction of mutations at specific sites in genes, that is, insertion, deletion or substitution of a certain length of nucleotide sequences in known DNA sequences. This method has been widely used in the study of gene expression regulation, gene structure and function, and protein properties. [0003] The most common method for generating SDM is Overlap extension PCR (OE-PCR)-mediated site-directed mutagenesis, also known as overlap extension PCR. This technology is a simple and rapid gene recombination technology for in vitro gene splicing of two or more gene fragments through the method of terminal complementarity and...

Claims

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Application Information

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IPC IPC(8): C12P19/34
Inventor 吴文芳安迎锋吕安国
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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