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Nucleic acid molecular cloning method and related kit based on homologous recombination

A homologous recombination and nucleotide technology, applied in the field of DNA recombination, can solve problems such as difficulty in distinguishing single nucleotide mutations, low conversion rate, and amplification of large fragments of genomic DNA

Inactive Publication Date: 2012-08-15
SHENZHEN ZHONGLIAN BIOLOGICAL TECH DEV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, existing cloning methods based on homologous recombination still have some problems, especially in the cloning of large fragments of genomic DNA from eukaryotes or the study of single nucleotide polymorphisms (SNPs) in human genomic DNA in the case of
For example, it is currently difficult to amplify large fragments of genomic DNA above 10kb by PCR
For in vivo homologous recombination, as it involves co-transformation or co-transfection of a PCR-amplified target DNA fragment with a vector DNA molecule into host cells, resulting in low transformation efficiencies
In addition, when conducting SNP research, it is difficult to distinguish whether the detected single nucleotide mutation is present in the genome itself or artificially introduced due to PCR amplification

Method used

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  • Nucleic acid molecular cloning method and related kit based on homologous recombination
  • Nucleic acid molecular cloning method and related kit based on homologous recombination
  • Nucleic acid molecular cloning method and related kit based on homologous recombination

Examples

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preparation example Construction

[0048] Preparation of extended linearized vector

[0049] According to this application, the first sequence and the second sequence are added to both sides of the linearized vector to prepare an extended linearized vector, wherein the first sequence contains a sequence homologous to the first end of the target DNA or its flanking sequence, and the second sequence The second sequence contains a sequence homologous to the second end of the target DNA or its flanking sequence, so that the linearized vector added with the first sequence and the second sequence on both sides can be connected with the target DNA through homologous recombination. The first sequence and the second sequence may be sequences homologous to the corresponding ends of the target DNA or sequences flanking them.

[0050] The so-called "homologous" means that two nucleotide sequences have a certain sequence identity (identity) or homology (homology), so that they can be linked together by homologous recombin...

Embodiment 1

[0092] The human DHRS4 gene cluster has three gene copies, namely DHRS4 (15.569bp), DHRS4L2 (about 35kb) and DHRS4L1 (also known as DHRS4X), among which the former two are highly homologous (90%-98%) and belong to fragments Replication (segmental duplication). The homology between DHRS4L1 and DHRS4 and DHRS4L2 was 77.8% and 77.7%, respectively. The high homology between the three genes limits the application of conventional molecular biology methods and brings difficulties to the sequencing of the DHRS4 gene (full length 15.569bp), that is, through the next-generation gene sequencing technology and gene chip capture technology (from Agilent and Nalgene) are difficult to carry out accurate sequencing and SNP research of this gene.

[0093] In this example, the homologous recombination of the DHRS4 gene into the p15A vector (Prutin Biotechnology (Beijing) Co., Ltd.) was mainly performed using an enzyme mixture containing RecE and RecT. In short, by using the 15-50bp sequences ...

Embodiment 2

[0122] According to the method described in Example 1, suitable PCR primers were designed, and the mouse TFIIA gene (transcription factor IIA, Transcription factor II A) with a length of about 30 kb was cloned from the cosmid LAWRIST7-mTFIIA (Gene Bridges GmbH) onto the p15A vector . The PCR products of the plasmids obtained from positive clones were verified by PstI restriction map. Figure 4 The enzyme digestion patterns of the PCR products obtained from some positive clones are shown. It can be seen from the figure that lanes 2-13 in Figure B are normal connections, while lane 14 is incorrect connections. According to statistics, the successful connection rate of cloning according to the method of the present application (the number of correctly connected clones / the number of detected positive clones) is about 65-70%.

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Abstract

The invention provides a nucleic acid molecular cloning method based on homologous recombination. According to the method provided by the invention, a target DNA is cloned to a vector by the homologous recombination through providing a linearizing vector with two ends respectively added with sequences(namely specific homologous arm of the target DNA) homologous with sequences at the two ends of the target DNA or the flanking sequence of the target DNA or utilizing a connecting section containing the specific homologous arm of the target DNA as well as the specific homologous arm of the vector (sequence homologous with the specific region of the vector). The nucleic acid molecular cloning method provided by the invention is especially suitable for the clone of large DNA section and polymorphism researches of mononucleotide. The invention further provides a related kit.

Description

field of invention [0001] This application relates to the field of DNA recombination technology. More specifically, this application relates to a nucleic acid molecular cloning method based on homologous recombination, its application and related kits. Background technique [0002] In molecular biology research and the biotechnology industry, there is always a need to clone the desired DNA molecule into a vector, especially at a specific location on the vector. [0003] The traditional method of cloning target DNA into a predetermined position in a vector such as a plasmid usually includes six major steps: (1) digesting the vector DNA with a restriction endonuclease and purifying the linearized vector; (2) using a small Cattle intestinal alkaline phosphatase (CIP) is used to process the linearized carrier to minimize the degree of self-circularization of the linearized carrier during connection; (3) PCR primers are used to amplify the target DNA by means of polymerase chain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63
CPCC12N15/902C12N15/1082C12N15/09C12N15/63C12N15/66
Inventor 于浩洋
Owner SHENZHEN ZHONGLIAN BIOLOGICAL TECH DEV
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