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Artificially designed plasmid pM5500 and application thereof in preparation of DNA marker

A plasmid, artificial technology, applied in DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of complicated preparation process and insufficiently compact plasmid structure, and achieve high amplification efficiency, stability and stability. Amplification efficiency, easy-to-estimate effects

Active Publication Date: 2020-09-25
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problems existing in the prior art, the present invention discloses a plasmid pM5500 and a construction method for the preparation of medium molecular weight DNA markers, which solves the problem of medium molecular weight DNA marker preparation at present The plasmid structure is not compact enough, and the preparation process is complicated and cumbersome

Method used

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  • Artificially designed plasmid pM5500 and application thereof in preparation of DNA marker
  • Artificially designed plasmid pM5500 and application thereof in preparation of DNA marker
  • Artificially designed plasmid pM5500 and application thereof in preparation of DNA marker

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Embodiment 1

[0024] like figure 1 , this embodiment provides a plasmid pM5500 for the preparation of medium molecular weight DNA markers, its sequence is shown in SEQ ID NO: 1, including high-copy replication origin sequence and ampicillin resistance gene coding sequence, and various artificially designed Combined DNA fragments. The plasmid contains 12 homogeneous restriction endonuclease recognition sites within a limited length, and can produce 1500, 1000, 750, 500, 250 and 100bp common DNA fragments in six medium-molecular-weight DNA markers by single enzyme digestion, And the mass ratio of various fragments is 3:2:3:1:1:1.

Embodiment 2

[0026] This example provides a method for constructing pM5500 and similar plasmids: select a suitable infrastructure plasmid, such as pUC19, pBR322, LITMUS28i, etc., introduce restriction enzyme sites into the vector infrastructure, and make the distance between the cutting points meet expected. Additional length fragments are subsequently introduced at the multiple cloning site of the vector. Finally, the plasmid contained one fragment of 1500bp, one fragment of 1000bp, two fragments of 750bp, one fragment of 500bp, two fragments of 250bp and five fragments of 100bp. These fragments can be derived from known plasmids, or amplified from DNA of other species, or obtained by chemical synthesis. Fragments as desired in the present invention were amplified from phage lambda and E. coli genomes. The designed plasmid is 5500bp in length and contains 12 recognition sites for a specific restriction endonuclease (12 BamH I recognition sites in this example). The total length of the ...

Embodiment 3

[0028] This embodiment provides a construction of pM5500 and similar vectors, and the construction of pM5000 is divided into three steps:

[0029] Step 1: Transformation of the basic plasmid backbone. The construction starts with a plasmid called pM3K, which is transformed based on the LITMUS28i plasmid, with a size of 3kb and a BamH I cutting site. like figure 2 As shown, a BamH I cutting point is introduced in the upstream and downstream of the ampicillin resistance gene of the backbone plasmid pM3K, so that the modified pM3K contains 3 BamH I cutting points, and three fragments of 1500, 1000, and 500 bp are produced after enzyme digestion. When introducing the BamH I cutting point, the pM3K plasmid was first used as a template to amplify, and the primers contained the BamH I recognition sequence, that is, the upstream and downstream 15-20nt sequences, and two DNA fragments with a length of 1033bp and 2034bp were obtained respectively. Then use Novizym's ClonExpress II On...

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Abstract

The invention discloses an artificially designed plasmid pM5500 and an application thereof in preparation of a DNA marker. The length of the plasmid is 5500 bp, the sequence of the plasmid is SEQ ID NO: 1, and the plasmid comprises a high-copy replication starting point sequence, an ampicillin resistance gene coding sequence and various artificially designed and combined DNA fragments. The plasmidcontains 12 restriction endonuclease recognition sites of the same kind within the limited length; six DNA fragments of 1500, 1000, 750, 500, 250 and 100bp suitable for medium molecular weight DNA marker preparation can be generated through single endonuclease digestion, and the mass ratio of the fragments is 3:2:3:1:1:1. The plasmid is compact and stable in structure, high in copy number and easy to prepare on a large scale, and the DNA maker developed on the basis of the plasmid can be widely applied to the fields of biological research and biotechnology.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to molecular biology, genetic engineering and biotechnology applications, in particular to an artificially created plasmid pM5500 and its application in DNA marker preparation. Background technique [0002] DNA marker is a mixture of DNA fragments of known size and quantity, commonly used in nucleic acid electrophoresis to indicate the size, shape and relative quality of unknown DNA samples. As one of the most basic biology and biotechnology research tools, DNAmarker has an irreplaceable role and application market. [0003] There are three main sources of DNA fragments currently used for DNA marker preparation, one is obtained by phage or plasmid digestion, the other is obtained by PCR amplification, and the third is obtained by chemical synthesis. Single-stranded or double-stranded short fragments (100bp or less) can be synthesized chemically, 100-5000bp can be obtained by plasmid digestio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66C12N15/11C12Q1/6806
CPCC12N15/63C12N15/66C12Q1/6806C12Q2565/125C12Q2521/301
Inventor 单丽伟范三红胡小平
Owner NORTHWEST A & F UNIV
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