Chemical modification method of DNA (Deoxyribonucleic Acid) polymerase

Abstract

The invention discloses a chemical modification method of DNA (Deoxyribonucleic Acid) polymerase and belongs to the technical field of biological detection. The chemical modification method comprises the following steps: I, carrying out self construction on Taq DNA polymerase extracted from engineering bacteria; II, preparing a chemical modifier, namely uniformly mixing solutions of EDC (Dichloroethane), NHS (N-Hydroxy Succinimide) and -COOH in a volume ratio of 1:9:5, carrying out a reaction for 25 minutes at 30-37 DEG C, and gently turning over and uniformly mixing the components up and down once every 5 minutes; III, carrying out Taq DNA polymerase modification, namely sufficiently mixing the modifier prepared in the step II with the Taq DNA polymerase of 3-6 times in volume, carrying out a reaction for 25 minutes at 30-37 DEG C, and gently turning over and uniformly mixing the components up and down once every 5 minutes; IV, preparing warm start Taq DNA polymerase, namely adding 1% BSA (Bovine Serum Albumin) of a same volume after modification is completed in the step III, sufficiently, gently and uniformly mixing, further adding 80% glycerinum of a same volume, and preserving in an environment of minus 20 DEG C, thereby obtaining the warm start Taq DNA polymerase. The modification mode of the Taq DNA polymerase disclosed by the invention is simple.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a chemical modification method of DNA polymerase. Background technique [0002] Polymerase chain reaction (PCR) is a technology invented by American scientist Kary BanksMulls in 1985 that can rapidly amplify specific genes or DNA sequences in vitro. The main principle of this Nobel Prize-winning technology is to use double-stranded DNA as a template, a synthetic nucleotide sequence as a primer, and dNTPs as a substrate to obtain a large number of gene copies through the catalysis of DNA polymerase. DNA polymerase, also known as DNA-dependent DNA polymerase, mainly plays a role in polymerization: that is, at the end of primer RNA-OH, dNTP is used as a substrate, and the instructions on the template DNA, that is, the pairing of A and T, C and G In principle, dNTPs are gradually and continuously added to the 3'-OH end of the elongating DNA molecule, and the elongating ...

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Problems solved by technology

[0005] At present, the preparation method of hot-start DNA polymerase is mainly through the transformation and modification of Taq DNA polymerase by utilizing the positioning of the functional region of Taq DNA polymerase. The main modification method is nucleic acid aptamer modification. combined with TaqDNA polymerase to block the activity of the enzyme at room temperature. This modification method involves gene technology. A high-affinity nucleic acid aptamer; specific antibody modification involves the basic principles and techniques of immunology. The modified TaqDNA polymerase is used as an antigen to immunize experimental animals to produce corresponding antibodies. After a series of screening, The antibody is prepared on a large scale by monoclonal antibody technology, and the protein biological activity of the antibody is used to inactivate and fall off at the high temperature of PCR reaction at 90°C to achieve the effect of PCR hot start. The principle of this modification technology is better than that of nucleic acid aptamers. The modification should be simple and easy to understand, but the production of specific antibodies requires a very long screening cycle, and at the same time, it is easy to cause great pain to the immunized animals, which is not well in line with the international experimental animal welfare

Acid anhydride modification mainly uses the reversible combination of maleic anhydride and citraconic anhydride to specific active amino acid groups to block its activity at room temperature, and the combination falls off at high temperature to achieve a hot start effect. This modification method is currently popularized by people. Agreed, the experimental cycle is shorter than the above two methods, but the cost of funds is still high, and the modification method is cumbersome

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