PCV2 strain infectious cloning construction kit and method based on in-vitro rolling circle replication

A technology of infectious cloning and rolling circle replication, applied in biochemical equipment and methods, cells modified by introducing foreign genetic material, viruses, etc., can solve the problems of poor template sensitivity, non-specific amplification interference, etc., and achieve simplified separation Process, the effect of ensuring virus specificity

Inactive Publication Date: 2016-08-31
HUNAN AGRICULTURAL UNIV
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Problems solved by technology

If the genome sequence of the virus is unknown, it can also be amplified by random primers, but the non-specificity of the primers will cause non-specific amplification interference of circular DNA such as the whole genome of other orbiviruses, which is also the sensitivity of this kind of RCA to templates Not as good as a drawback of the PCR method

Method used

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  • PCV2 strain infectious cloning construction kit and method based on in-vitro rolling circle replication
  • PCV2 strain infectious cloning construction kit and method based on in-vitro rolling circle replication
  • PCV2 strain infectious cloning construction kit and method based on in-vitro rolling circle replication

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Embodiment Construction

[0027] first part:

[0028] 1.1 PCV2 genome MPRCA

[0029]MPRCA was performed on PCV2 DNA (GenBank accession number KJ867555) and pUC19 plasmid (2686bp, MPRCA positive control). The system is as follows: Add 4 μL of template or 0.5 μL of pUC19, 20 μL of sample buffer (10 mM Tris-HCl, 0.1 mMEDTA) into the PCR tube, put it into an ABI 2700 PCR instrument at 95 ° C for 3 min, cool to 4 ° C, and then add 20 μL reaction buffer 【330mM Tris-acetate (pH 7.9at 37℃), 100mMMg-acetate, 660mM K-acetate, 1% (v / v) Tween 20, 10mM DTT, 30pmol PCV2-specific upstream primer 5'-CCATATGAAATAAATTACTGAG- 3'(SEQ ID NO.1), PCV2-specific upstream primer 5'-CAGCGCACTTCTTTCGTTTTTCAG-3'(SEQ ID NO.2) and random primer 5'-NNNNNN-3'(SEQ ID NO.3) and 0.8μL phi29DNA polymerization The enzyme (purchased from Thermo Scientific Company) was reacted at 30°C for 18h, then at 65°C for 10min, and finally cooled to 4°C, and the final product of the MPRCA reaction was stored at -20°C.

[0030] 1.2 Digestion and liga...

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Abstract

The invention discloses a PCV2 strain infectious cloning construction kit and method based on in-vitro rolling circle replication. The PCV2 strain infectious cloning construction kit based on in-vitro rolling circle replication contains the following primers: a PCV2 specific upstream primer: 5'-CCATATGAAATAAATTACTGAG-3' (SEQ ID NO. 1); a PCV2 specific downstream primer: 5'-CAGCGCACTTCTTTCGTTTTCAG-3' (SEQ ID NO. 2); and a random primer: 5'-NNNNNN-3' (SEQ ID NO. 3). The PCV2 strain infectious cloning method comprises the following steps: performing multi-primer rolling circle replication on PCV2 DNA by using the abovementioned primers to obtain a PCV2 whole genome; separating the whole genome through single enzyme digestion, purifying the separated whole genome, and connecting the purified whole genome; performing cyclizing once again; constructing PCV2 strain infectious cloning.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit and method for constructing infectious clones of PCV2 (porcine circovirus type 2) strains based on in vitro rolling circle replication. Background technique [0002] Rolling-circle amplification (RCA) is a DNA amplification technique established in 1998 that can be used to amplify circular DNA templates at constant temperature. This technology is a nucleic acid amplification technology based on the rolling circle replication method of circular DNA molecules of pathogenic microorganisms in nature. At present, RCA can use polymerases such as Phi29, Bst and Phage T7 to complete rolling circle replication in vitro. These enzymes have strong persistence and strand displacement capabilities, and can meet the requirements of the RCA amplification mechanism. The polymerase can continuously extend the single-stranded circular DNA template with the help of primers. After com...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C12N5/10
CPCC12N15/11C12N7/00C12N15/10C12N2750/10052C12Q2531/125
Inventor 王乃东王占峰杨毅王爱兵邓治邦杨林张颜朱哲
Owner HUNAN AGRICULTURAL UNIV
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