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Method and vector for cloning target dna fragment

A technology of fragments and vectors, applied in the field of genetic engineering, can solve problems such as high time cost

Active Publication Date: 2022-02-01
NANTONG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that it needs repeated genetic operations, and the time cost is relatively large.

Method used

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  • Method and vector for cloning target dna fragment
  • Method and vector for cloning target dna fragment
  • Method and vector for cloning target dna fragment

Examples

Experimental program
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Embodiment 1

[0068] Example 1, the method of directly cloning the act gene cluster from Streptomyces coelicolor using CRISPR-Cas9 technology.

[0069] Reagents and carriers used, etc.

[0070] pCRISPR-Cas9 (ACS Synth. Biol., 2015, 4(9), pp. 1020–1029; Genbank accession number KR011749, image 3 ) vector from the laboratory of Professor Tilmann Weber at the Technical University of Denmark. The vector can also be self-synthesized according to the vector sequence published by Genbank.

[0071] The Streptomyces coelicolor M145 strain is a widely used type strain for Streptomyces research. The Streptomyces coelicolor M145 strain of the present invention is derived from the ATCC strain collection (deposit number ATCC BAA-471).

[0072] In this example, the act synthetic gene cluster was directly cloned from Streptomyces coelicolor M145 strain. Include the following steps:

[0073] 1. Transform the pCRISPR-Cas9 vector, delete the SG5 replicon (located at 9627-11069bp of the vector), so that ...

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Abstract

The present invention provides a method and a carrier for cloning a target DNA fragment, the method comprising: identifying a CRISPR-Cas9 recognition site located on one side of the target DNA fragment, and an integration site and a first site respectively located on both sides of the target DNA fragment A recombination site, the first recombination site is located between the target DNA fragment and the CRISPR-Cas9 recognition site; the integration site, the recognition site and the first recombination site are cloned into the vector, and the The spacer sequence is integrated into the 5' end of the sgRNA sequence on the vector; the vector is integrated into the genetic material where the target DNA fragment is located by homologous recombination; the expression of the Cas9 coding sequence of the positive clone is induced, and the cutting Homologous recombination occurs between the two first recombination sites at the two ends of the generated fragment; the plasmid carrying the target DNA fragment is screened. The method of the invention has simple steps, low cost and high efficiency, and is especially suitable for cloning large target DNA fragments from organisms.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method and a carrier for cloning target DNA fragments. Background technique [0002] Genome editing methods based on the CRISPR (Clustered Regularly InterspacedSmall Palindromic Repeats) / Cas9 system have profoundly changed the research methods and research content in the field of life sciences. The CRISPR / Cas9 system is one of the widely existing adaptive immune systems in prokaryotes and is used to deal with the invasion of exogenous nucleic acid molecules, such as degrading phage-derived nucleic acid molecules, and degrading exogenous plasmids transformed into cells. After the cell is invaded for the first time, part of the exogenous DNA sequence is captured and regularly integrated in the form of spacers to become CRISPR sequences. In order to accurately distinguish self and foreign DNA molecules, there is a PAM (Protospacer Adjacent Motif) motif 5'-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/66C12N15/65C12N15/63
CPCC12N9/22C12N15/63C12N15/65C12N15/66C12N2810/10
Inventor 苗靳
Owner NANTONG UNIVERSITY