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A method for high-accuracy site-directed gene knockout in yeast

A high-accuracy, gene-knock-out technology, applied in the field of genetic engineering, achieves the effects of simple technical methods, reduced labor, and reduced material expenditures

Active Publication Date: 2020-05-08
ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The key innovation in the design of the present invention - the structure of "linear 3' protruding long sticky end double-stranded DNA" and its application have not been reported yet

Method used

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  • A method for high-accuracy site-directed gene knockout in yeast
  • A method for high-accuracy site-directed gene knockout in yeast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Step 1: 1000 bp homologous sequence cloning of upstream (sequence A1) and downstream (sequence B1) of tor2 gene.

[0027] Cloning by polymerase chain reaction (PCR), the DNA polymerase used is the high-fidelity DNA polymerase (Phusion DNA Polymerase) produced by NEB, the amplification template is the genomic DNA of Saccharomyces cerevisiae gim2. Design and synthesize oligonucleotides, PPF1, PPR1, PTF1 and PTR1, the sequence information is as follows:

[0028] PPF1:5'-GACCATGATTACGCCAAGCTTGC TTTTCATATGGGGAAAGTAA-3'

[0029] PPR1:5'-GAAGATGCGGCCGCTGTTACAAGAAAGCGGAGGGAGAAGA-3'

[0030] PTF1:5'-GTAACAGCGGCCGCATCTTCCTTCTTCCGCAATTCCAGCAG-3'

[0031] PTR1:5'-GACCTGCAGGCATGCAAGCTTGG TTTTAATGTATTGAAAATCA-3'

[0032] Primers PPF1 and PPR1 are used to clone the 1kb region upstream of tor2, and PTF1 and PTR1 are used to clone the 1kb region downstream of tor2. The PCR reaction system is as follows:

[0033] 5×HF reaction buffer (Mg 2+ Concentration is 7.5mM) 10μL, ddH 2 O 31....

Embodiment 2

[0056] Step 1: 100 bp homologous sequence cloning of upstream (sequence A2) and downstream (sequence B2) of tor2 gene.

[0057] The cloning method is the same as in Example 1, the primers are PPF2, PPR1, PTF1 and PTR2, and the sequence information is as follows:

[0058] PPF1:5'-GACCATGATTACGCCAAGCTTGC TTTTCATATGGGGAAAGTAA-3'

[0059] PPR2:5'-GAAGATGCGGCCGCTGTTAC AAAGGGAAAA TATACCGGGT-3'

[0060] PTF2: 5'-GTAACAGCGGCCGCATCTTC GTAACGTCACGCTCGGAACT-3'

[0061] PTR1:5'-GACCTGCAGGCATGCAAGCTTGG TTTTAATGTATTGAAAATCA-3'

[0062] Primers PPF1 and PPR2 are used to clone the upstream 100bp region of tor2, and PTF2 and PTR1 are used to clone the downstream 100bp region of tor2. The PCR reaction system is the same as in Example 1 except that primer PPR2 replaces PPR1 and PPF2 replaces PPF1. The PCR amplification conditions were the same as in Example 1 except that the extension time was changed to 20 sec, and the PCR product purification method was the same as in Example 1.

[0063] S...

Embodiment 3

[0075] Step 1: 500 bp homologous sequence cloning of upstream (sequence A3) and downstream (sequence B3) of tor2 gene.

[0076] The cloning method is the same as in Example 1, the primers are PPF3, PPR1, PTF1 and PTR3, and the sequence information is as follows:

[0077] PPF1:5'-GACCATGATTACGCCAAGCTTGC TTTTCATATGGGGAAAGTAA-3'

[0078] PPR3:5'-GAAGATGCGGCCGCTGTTAC TATATATTTA TTACCGTCAT-3'

[0079] PTF3: 5'-GTAACAGCGGCCGCATCTTC CGGTAACAGGACAACAGCCA-3'

[0080] PTR1:5'-GACCTGCAGGCATGCAAGCTTGG TTTTAATGTATTGAAAATCA-3'

[0081] Primers PPF1 and PPR3 are used to clone the 500bp region upstream of tor2, and PTF3 and PTR1 are used to clone the 500bp region downstream of tor2. The PCR reaction system is the same as in Example 1 except that primer PPR3 replaces PPR1 and PPF3 replaces PPF1. The PCR amplification conditions were the same as in Example 1 except that the extension time was changed to 30 sec, and the PCR product purification method was the same as in Example 1.

[0082] S...

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Abstract

The invention provides a method for realizing high-accuracy fixed-point gene knockout in yeast. After obtaining the upstream and downstream sequences of the target knockout gene by the method of polymerase chain reaction or whole gene synthesis, the upstream and downstream sequences are reversed from the original gene. Direction connection, and inserted into the carrier plasmid to construct a circular cloning vector, linearize the circular cloning vector to obtain linear double-stranded DNA, and treat the linear double-stranded DNA with 5'→3' exonuclease to form Transform yeast cells after linear 3' overhanging long sticky double-stranded DNA. The invention realizes high-accuracy fixed-point gene knockout in yeast by means of a special exogenous DNA structure type. The accuracy rate of targeted gene knockout can reach 45-92%, which can greatly reduce the workload of yeast targeted gene knockout, and has great application potential in the research of yeast functional genes and the construction of genetic engineering strains.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and mainly relates to a method for realizing high-accuracy fixed-point gene knockout in yeast. Background technique [0002] Yeast is a general term for a class of unicellular eukaryotic microorganisms in the fungal kingdom. There are more than 1,500 identified species, accounting for about 1% of the identified fungal species. The diameter of yeast cells is about 2-6 μm, the length is 5-30 μm, and some are longer. The individual shapes are spherical, oval, elliptical, columnar and sausage-shaped, etc., and are widely distributed in nature. Most yeasts can be isolated in sugar-rich environments, such as the surface of some fruits, fermented grains, plant secretions, and even inside insects. Most yeasts like to grow in acidic, moist and sugary environments, and can survive in both aerobic and anaerobic environments. They are facultative anaerobes. [0003] Yeast is the earliest microorganism us...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12R1/865
CPCC12N15/81C12N2800/102C12N1/185C12R2001/865
Inventor 柳永刘士旺鲍文娜
Owner ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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