5 results about "Klenow fragment" patented technology

The present application relates to the application of Klenow mutant fragments in the synthesis of nucleic acids, and belongs to the technical field of biological modification and synthesis.The present application provides the application of Klenow mutant fragments with an amino acid sequence as shown in SEQ ID NO.8 or SEQ ID NO.14 in the synthesis of nucleic acids.The mutant protein F762A expressed by the Klenow mutant fragments of the present application can efficiently synthesize deoxyribonucleic acid, ribonucleic acid, deoxyribonucleic acid analogs and ribonucleic acid analogs with different lengths of DNA templates and DNA / RNA primers at 37 DEG C, and has a wide synthesis range and universality.

The invention relates to a method for detecting DNA (deoxyribonucleic acid) repair enzyme on demand by remolding and regulating Cas12a activity through PAM (polyacrylamide). The method comprises the following steps: step 1, annealing a hairpin probe HP1 in an annealing buffer solution to form a hairpin structure; 2, DNA repair enzyme is added into a reaction solution, the reaction solution comprises a hairpin probe HP1, APE1, klenow fragment DNA polymerase, a dNTP mixed solution, Cas12a, crRNA, reporter molecules and an NEBuffer 2 reaction buffer solution, then incubation is conducted, and the purposes of regulating and controlling the activity of the Cas12a and detecting the DNA repair enzyme are achieved. According to the invention, high tense and hierarchical regulation and control of Cas12a trans-cleavage activity are realized, and a universal sensing platform (namely a CRISPR switch) is convenient to construct and is used for detecting a plurality of DNA (deoxyribonucleic acid) repair enzymes at high sensitivity as required.

A method for preparing a 5′-end ligation-based ssDNA-specific sequencing (Liss-seq) library is provided. The method includes: (a) treating a DNA sample to be tested that includes single-stranded DNA and double-stranded DNA with Klenow Fragment (3′→5′exo−) DNA polymerase to fill in 5′ ends of the double-stranded DNA to obtain a first reaction product; (b) conducting a 3′ end tail addition reaction on the first reaction product to obtain a second reaction product; (c) ligating the second reaction product with a renatured 5′ hairpin adaptor to obtain a ligation product; a structure of 5′ hairpin adaptor being: 5′-overhang-random base region-first stem region-loop region-second stem region-3′, the first stem region and the second stem region forming a double-strand through a renaturation treatment; and (d) amplifying the ligation product to obtain the Liss-seq library, a length of the overhang being within a range of 0-20 nt.

Provided are a modified Klenow fragment and an application thereof; specifically provided is a modified Klenow fragment, wherein at least one position or functionally equivalent position among F762, A842, I709 and P603 in the amino acid sequence of the modified Klenow fragment contains at least one amino acid substitution mutation. The modified Klenow fragment has higher DNA polymerase activity than a wild-type Klenow fragment and can be applied to sequencing.

The application discloses an enzyme composition for low-preference DNA fragmentation and a reaction solution thereof. The enzyme composition comprises an endonuclease, a nucleotide kinase and a DNA polymerase, the endonuclease comprises any one of DNase I, heat-stable high-salt-tolerant endonuclease HL-SAN or endonuclease IV or a combination of at least two of them, the nucleotide kinase comprises T4 polynucleotide kinase, and the DNA polymerase comprises any one of Taq DNA polymerase, a Klenow fragment of DNA polymerase I or T4 DNA polymerase or a combination of at least two of them. The application develops a one-tube method for genome fragmentation, end repair and A-tailing module, including an enzyme composition and a reaction solution thereof. In view of the problems and deficiencies of the current enzyme library construction, the components are optimized and explored, and a suitable connection module is combined, so that the finally obtained product has low preference, the output library has higher uniformity, and has a wide application prospect in second-generation sequencing.