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Method for realizing high-accuracy gene homologous cloning in yeast

A high-accuracy, yeast-based technology, applied in the field of genetic engineering, achieves the effects of simple technical methods, high accuracy, and reduced labor

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

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  • Method for realizing high-accuracy gene homologous cloning in yeast
  • Method for realizing high-accuracy gene homologous cloning in yeast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Step 1: 1000 bp homologous sequence cloning of upstream (sequence A1) and downstream (sequence B1) of tor2 gene.

[0028] 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:

[0029] PPF1:5'-GACCATGATTACGCCAAGCTTGCAAGAAAGCGGAGGGAGAAGA-3'

[0030] PPR1:5'-GAAGATGCGGCCGCTGTTAC TTTTCATATGGGGAAAGTAA-3'

[0031] PTF1: 5'-GTAACAGCGGCCGCATCTTC TTTTAATGTATTGAAAATCA-3'

[0032] PTR1:5'-GACCTGCAGGCATGCAAGCTTGG CTCTTCCGCAATTCCAGCAG-3'

[0033] 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:

[0034] 5×HF reaction buffer (Mg 2+ Concentration is 7.5mM) 10μL, ddH 2 O 31...

Embodiment 2

[0058] Step 1: 100 bp homologous sequence cloning of upstream (sequence A2) and downstream (sequence B2) of tor2 gene.

[0059] 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:

[0060] PPF2: 5'-GACCATGATTACGCCAAGCTTGC AAAGGGAAAA TATACCGGGT-3'

[0061] PPR1:5'-GAAGATGCGGCCGCTGTTAC TTTTCATATGGGGAAAGTAA-3'

[0062] PTF1: 5'-GTAACAGCGGCCGCATCTTC TTTTAATGTATTGAAAATCA-3'

[0063] PTR2:5'-GACCTGCAGGCATGCAAGCTTGGGTAACGTCACGCTCGGAACT-3'

[0064] Primers PPF2 and PPR1 were used to clone the upstream 100bp region of tor2, and PTF1 and PTR2 were used to clone the downstream 100bp region of tor2. The PCR reaction system was the same as in Example 1 except that primer PPF2 replaced PPF1 and PPR2 replaced PPR1. 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.

[0065...

Embodiment 3

[0078] Step 1: 500 bp homologous sequence cloning of upstream (sequence A3) and downstream (sequence B3) of tor2 gene.

[0079] 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:

[0080] PPF3:5'-GACCATGATTACGCCAAGCTTGC TATATATTTA TTACCGTCAT-3'

[0081] PPR1:5'-GAAGATGCGGCCGCTGTTAC TTTTCATATGGGGAAAGTAA-3'

[0082] PTF1: 5'-GTAACAGCGGCCGCATCTTC TTTTAATGTATTGAAAATCA-3'

[0083] PTR3:5'-GACCTGCAGGCATGCAAGCTTGGCGGTAACAGGACAACAGCCA-3'

[0084] Primers PPF3 and PPR1 are used to clone the 500bp region upstream of tor2, and PTF1 and PTR3 are used to clone the 500bp region downstream of tor2. The PCR reaction system is the same as in Example 1 except that primer PPF3 replaces PPF1 and PPR3 replaces PPR1. 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.

[0085] St...

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Abstract

The invention provides a method for realizing high-accuracy gene homologous cloning in saccharomycetes, comprising the steps: obtaining the upstream sequence and downstream sequence of a target cloning gene by polymerase chain reaction or full gene synthesis, connecting the upstream sequence and downstream sequence and inserting into a carrier plasmid for constructing an annular cloning vector, performing linearization treatment on the annular cloning vector to obtain linear double-stranded DNA, and performing 5' ->3' exonuclease treatment on the linear double-stranded DNA to form linear 3' protruding long cohesive end double-stranded DNA and converting a saccharomycete cell. The method makes use of a special extraneous DNA structure type and realizes high-accuracy gene homologous cloning in saccharomycetes. The homologous gene cloning accuracy can reach 50-95%, and the method can greatly reduce the homologous gene cloning workload of saccharomycetes, and has great application potential in study on saccharomycete functional genes, gene engineering strain construction, and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and mainly relates to a method for realizing high-accuracy gene homologous cloning 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 used...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N15/90
CPCC12N15/81C12N15/905
Inventor 柳永刘士旺鲍文娜
Owner ZHEJIANG UNIVERSITY OF SCIENCE AND TECHNOLOGY
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