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Genome-wide construction of schizosaccharomyces pombe heterozygous deletion mutants containing gene-specific barcodes by the methods of 4-round serial or block pcr, or total gene synthesis thereof

A fission yeast, barcode technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of low success rate, low homologous recombination rate of fission yeast, difficulty in strain preparation, etc.

Active Publication Date: 2011-04-13
KOREA RES INST OF BIOSCI & BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such a low success rate in fission yeast suggests that strain preparation is very difficult due to the lower rate of homologous recombination in fission yeast than in budding yeast

Method used

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  • Genome-wide construction of schizosaccharomyces pombe heterozygous deletion mutants containing gene-specific barcodes by the methods of 4-round serial or block pcr, or total gene synthesis thereof
  • Genome-wide construction of schizosaccharomyces pombe heterozygous deletion mutants containing gene-specific barcodes by the methods of 4-round serial or block pcr, or total gene synthesis thereof
  • Genome-wide construction of schizosaccharomyces pombe heterozygous deletion mutants containing gene-specific barcodes by the methods of 4-round serial or block pcr, or total gene synthesis thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Construction of deletion cassettes by four rounds of serial PCR or blocking PCR

[0093] Deletion cassettes can be constructed in two different PCR techniques: four rounds of serial PCR and blocking PCR. The two PCR-based methods were pre-prepared Kan r Common among barcode modules. The base sequence of KanMX4 is well known in the art and will not be specifically described. Primary PCR was performed with a pair of 70-mer 5′ and 3′ barcode primers, each containing a gene-specific barcode, and KanMX4 served as a template to prepare Kan r Barcode module ( figure 2 ). After electrophoresis on an agarose gel stained with ethidium bromide (EtBR), the Kan thus prepared was extracted from the gel by excision under UV light. r Barcode module.

[0094] The four rounds of serial PCR used to construct the deletion cassette were similar to the conventional PCR used to construct gene-targeted budding yeast or fission yeast mutants, but differed by the addition of ...

Embodiment 2

[0096] Example 2. Construction of deletion cassettes by gene synthesis

[0097] Even the PCR technique described above failed to generate gene-targeted mutants up to 4% of all genes. The reason is because the high content of adenine (A) and thymine (T) in the promoter and terminator of the gene does not allow primer sites for conventional PCR. Taking this issue into consideration, a gene synthesis method was utilized. The present invention is the first to use gene synthesis in constructing deletion cassettes and making deletion fission yeast mutants.

[0098] The deletion cassette was designed to consist of three fragments: 1) 5' chromosome homology region and 5' barcode with 5' common linker sequence (common likersequence), 2) kanamycin resistance gene (Kan 1 ), and 3) 3' barcodes and 3' chromosomal homology regions with 3' common linker sequences. To join the three fragments into a row, overlapping junction oligonucleotide sequences were arranged between the fragments t...

Embodiment 3

[0101] Example 3: Transformation of deletion cassettes and identification of gene-targeted mutants

[0102] Using the lithium acetate method, the deletion cassettes prepared by Examples 1 and 2 were transformed into diploid S. ) (Moreno et al., Methods Enzymol. 194:795-823). Spread the transformed strains on YES agar plates and culture them at 30°C for 3-4 days to form colonies. Colony PCR was performed to check if the desired gene was targeted. Pick a small number of cells from the edge of the growing colony on the agar plate and suspend them in deionized water. A portion of the suspension was subjected to PCR with a pair of primers. Such as Figure 6 Colony PCR was performed using a pair of CP5 and CPN1 or CPN10 for the 5' side of the deletion cassette for insertion into the chromosome, and a pair of CP3 and CPC1 or CPC3 for the 3' side of the deletion cassette as indicated. CP5 and CP3 are located at 100-200 bp upstream and downstream of the homologous regions of the...

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Abstract

A method comprising transforming Schizosaccharomyces pombe with a deletion cassette, constructed by four-round serial PCR, block PCR or total gene synthesis, containing a homologous recombination site is provided for preparing gene-targeted heterozygous deletion Schizosaccharomyces pombe. Also provided are gene-targeted hetero2ygous deletion Schizosaccharomyces pombe mutants prepared by the method, and a library of gene-targeted heterozygous deletion Schizosaccharomyces pombe mutants. Further, the library is useful in constructing a method and a kit for screening a drug's modes of action.

Description

technical field [0001] The present invention relates to a method for systematically preparing gene-targeted heterozygous deletion mutant Schizosaccharomyces pombe strains. More specifically, the present invention relates to a method for producing a gene-targeted heterozygous deletion of S. pombe by transforming S. pombe with a deletion cassette consisting of 4 rounds of sequential PCR, blocking PCR or Whole gene synthesis construct, containing homologous recombination sites. Meanwhile, the present invention relates to a gene-targeted heterozygous deletion S. pombe mutant and a library of gene-targeted heterozygous deletion S. pombe mutants prepared by the method. Furthermore, the present invention relates to methods and kits for screening libraries for the mode of action of drugs. Background technique [0002] In 2006, the worldwide pharmaceutical market was reported to be worth 643 billion USD, and Korean pharmaceutical products accounted for a 1,1472.8 billion won share ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/00
CPCC12N15/1034C12N1/16C12N1/18C12N15/00C12N15/09C12N1/00C40B30/00C40B40/02C40B50/00
Inventor 许光来金东旭元美善俞香淑金东燮朴翰浯郑璟淑张荣珠南美英韩尚助崔信正白昇泰金炯培许京善李惠美李慜镐朴助英
Owner KOREA RES INST OF BIOSCI & BIOTECH
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