4 results about "Rolling circle replication" patented technology

The invention belongs to the technical field of gene engineering, and relates to a plasmid of bacillus, an artificial recombinant vector as well as a construction method and application thereof, the plasmid is called pBso for short, and the nucleotide sequence of the plasmid is shown as SEQ ID NO.1. In a plasmid sequence, a protein coded by an open reading frame is a replication element Rep protein of a gram-positive bacterium rolling circle replication type plasmid, and the nucleotide sequence of the protein is shown as SEQ ID NO.2. The artificial recombinant vector is constructed by carrying out gene recombination on plasmids of the bacillus. The construction method of the artificial recombinant vector comprises the following steps: cloning a replication element Rep protein by using a molecular cloning means to construct the artificial recombinant vector with neomycin resistance. The replication element Rep protein of the plasmid pBso of the bacillus can be used for constructing an artificial recombinant vector, stably exists in the bacillus subtilis and the bacillus amyloliquefaciens and is subjected to replication and passage, and the steps of obtaining the engineering bacillus are simplified.

The invention relates to the technical field of phytopathogen molecule detection, and discloses a rolling circle replication-based high-sensitivity fusarium pseudograminearum detection method which comprises the following steps: extracting a to-be-detected genome of a sample; preparing closed-loop DNA (deoxyribonucleic acid); carrying out mixed reaction on the closed-loop DNA and exonuclease to obtain a purified circular DNA template; mixing the purified annular template, a rolling circle amplification primer, Phi29 DNA polymerase, dNTPs and a buffer solution, and carrying out rolling circle replication to obtain super-long single-stranded DNA; and mixing the rolling circle replication product, Cas12b protein, sgRNA, an ssDNA fluorescence report probe and a buffer solution, and carrying out trans-cleavage to release a fluorescence signal. According to the invention, the necessary in-vitro transcription step in the traditional isothermal amplification-CRISPR coupling technology is eliminated, the detection time is shortened, and meanwhile, the reagent cost and the operation error risk are reduced.

The invention relates to the technical field of hydrogel preparation, and discloses a preparation method of DNA hydrogel, which comprises the following steps: a, using plasmid as a template, and melting double strands of the plasmid template through alkaline denaturation; b, adding a random primer, enabling the plasmid template to be combined with the random primer in a renaturation process, and producing a large amount of double-stranded DNA with an ultra-long chain length and a high-branch topological structure as a DNA hydrogel precursor through hyper-branched rolling circle replication amplification; c, adding a dehydrating agent into the DNA hydrogel precursor solution, and realizing self-assembly by utilizing dense physical entanglement among double-stranded DNA molecules under the induction of the dehydrating agent to form a DNA aggregate; and d, collecting the DNA aggregate, drying the DNA aggregate to remove the residual dehydrating agent, resuspending the DNA aggregate in a buffer solution to enable the DNA aggregate to absorb water and swell, and finally forming the DNA hydrogel with a stable structure. According to the invention, the design and preparation process of the DNA hydrogel can be greatly simplified, the availability of the DNA hydrogel is improved, and the transformation of the DNA hydrogel from basic research to practical application is promoted.

The invention discloses a method for determining the stability of polymerase. The invention provides a method for detecting the stability of DNA (deoxyribonucleic acid) polymerase. The stability of the DNA polymerase is detected according to the principle that the DNA polymerase is reversibly inactivated and then activated. According to the scheme, operation is easy and convenient, the requirement for raw materials needed for amplification is low, radioactive isotopes, fluorescent markers and other fluorescent components do not need to be added in the amplification process, and pollution and interference caused by fluorescence are eliminated. The method is more suitable for reaction conditions of reagent development of high-throughput sequencing, whole genome amplification, isothermal amplification, rolling circle replication and the like, is more suitable for judgment of enzyme stability, provides reference for selection of modified enzyme or judgment of use conditions of specific enzyme, is convenient and time-saving, and reduces expenditure.