8 results about "Exochitinase activity" patented technology

The invention discloses a Taq DNA polymerase mutant with improved excision activity and application thereof, and belongs to the technical field of enzyme engineering. The invention aims to solve the technical problems of low probe cutting efficiency, weak fluorescence signal, limited detection sensitivity (especially for low-abundance targets), low reaction speed (high Ct value) and reduced specificity caused by insufficient 5 '-> 3' excision enzyme activity of wild Taq DNA polymerase in TaqMan probe method qPCR (quantitative polymerase chain reaction). According to the technical scheme, the Taq DNA polymerase mutant with the high 5 '-3' exonuclease activity is provided, the amino acid sequence of the Taq DNA polymerase mutant is shown as SEQ ID NO.2, the exonuclease activity of the mutant is improved by 77%, the cutting speed of a TaqMan probe is greatly increased, accumulation of fluorescence signals is accelerated, the Ct value is reduced, and the detection time is remarkably shortened.

The application discloses a hairpin probe for detecting DNA polymerase exonuclease activity and a preparation method and application thereof. The hairpin probe comprises a region a, a region b, a region c, a region d, a region e, a region f and a region g from the 5' end in sequence, wherein the region a and the region c are reversely complementary to each other, the 5' end of the region a is marked with a first fluorescent group, and the 3' end of the region a is marked with a first quenching group; the region e and the region g are reversely complementary to each other, the 5' end of the region g is marked with a second quenching group, and the 3' end of the region g is marked with a second fluorescent group; the first fluorescent group and the second fluorescent group are different from each other. The hairpin probe can be used for detecting the 3'-5' and 5'-3' exonuclease activity of the DNA polymerase separately or simultaneously in a single system, and has the advantages of simple operation steps, high throughput, good specificity, avoidance of radioactive pollution and excellent application prospect.

The invention belongs to the technical field of biology, and particularly relates to application of enzyme in isothermal nucleic acid amplification, a mixture, a kit and a detection method, in particular to application of human AP endonuclease 1 (hereinafter referred to as hAPE1) in the isothermal nucleic acid amplification technology. HAPE1 is applied to a multi-enzyme synergistic isothermal real-time amplification system such as RAA, RPA, ERA and MIRA; the fluorescent probe containing the AP site is efficiently cut by utilizing the high AP incision enzyme activity of hAPE1, so that real-time detection is realized; meanwhile, by virtue of the extremely low excision enzyme activity, the completeness of a DNA amplification product is maintained to the maximum extent, so that subsequent analysis and application are facilitated. While efficient real-time detection is ensured, it is ensured that an amplification product can be used for follow-up analysis, and therefore the application range of the technology is widened.

The invention belongs to the technical field of biology, and relates to a phi29 DNA polymerase mutant losing excision enzyme activity as well as a preparation method and application of the phi29 DNA polymerase mutant. According to the present invention, at least one amino acid residue in the 54th site, the 55th site, the 90th site, the 96th site, the 102th site, the 140th site, the 163th site, the 203th site, the 212th site, the 277th site, the 284th site, the 327 site, the 439th site, the 502th site, the 505th site, the 534th site and the 552th site in the phi29DNA polymerase amino acid sequence are modified to generate the novel protein having low excision enzyme activity but maintaining the polymerase activity, such that the problem possibly caused by the too high excision enzyme activity in the practical application of the phi29DNA polymerase is successfully solved; and the stability and the reliability in DNA amplification, sequencing and other molecular biology experiments are improved.

The application discloses a method for regulating catalytic behavior of 3'->5' exonuclease based on deoxyribose configuration adjustment and application thereof, and belongs to the technical field of nucleic acid chemistry, biological catalytic regulation and molecular engineering. In view of the problems of poor position accuracy and unpredictable effect of the existing nucleic acid enzyme regulation method, a hydrazine compound containing an NH-NH2 functional group is used to modify and reduce an AP site in DNA, so that the deoxyribose of the AP site is changed from a closed ring configuration to a stable open chain configuration. The DNA after the AP site is modified is incubated with a nucleic acid enzyme with 3'->5' exonuclease activity, so that the nucleic acid enzyme is specifically catalyzed to stop at a nucleotide downstream of the modified AP site, and the catalytic behavior of the nucleic acid enzyme is accurately and controllably regulated with single nucleotide resolution. The application does not need to rely on steric hindrance or enzyme molecular structure modification, the regulation effect is stable and predictable, and the application can be widely applied to high-resolution DNA damage positioning, controllable DNA assembly and DNA oxidative damage level detection.

The invention relates to a nested PCR (Polymerase Chain Reaction) method and application, and a targeted detection library building method improved based on the nested PCR library building method. According to the method, the nucleic acid cleavage activity of USER enzyme is utilized, and Taq DNA polymerase or an enzyme with both DNA polymerization activity and 5 '-3' excision enzyme activity is combined, so that a primer removal method is established and is used for replacing a purification step between a first round of PCR and a second round of PCR, and the problem of cross contamination between samples caused by purification operation of nested PCR is successfully solved. Meanwhile, the method can effectively remove non-specific amplification products and improve the specificity of targeted amplification, the experimental process is simpler and more convenient, and the nested PCR method can be applied to a targeted library building platform and a sequencing platform.

The application discloses a Taq enzyme mutant, a preparation method and application thereof. In the application, the Taq enzyme mutant comprises a mutant of an amino acid sequence shown in SEQ ID NO: 2, wherein the amino acid sequence is mutated at the following group of sites: P40, L125, G200, A335, G499, E634 and F769. The Taq enzyme mutant provided by the application has high amplification activity, more amplification products can be obtained compared with wild-type Taq enzyme under the same number of PCR cycles, and is resistant to whole blood and high salt and has low 5'-3' exonuclease activity.

The present invention relates to the technical field of biology and provides a phi29 DNA polymerase mutant lacking exonuclease activity, a preparation method therefor and a use thereof. By modifying at least one of amino acid residues at positions 54, 55, 90, 96, 102, 140, 163, 203, 212, 277, 284, 327, 439, 502, 505, 534, and 552 in an amino acid sequence of a phi29 DNA polymerase, a protein having reduced exonuclease activity and retained polymerase activity is generated, thereby solving problems possibly caused by excessive exonuclease activity during the actual application of phi29 DNA polymerases, and improving the stability and reliability of phi29 DNA polymerases in DNA amplification, sequencing, and other molecular biology experiments.