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CN-121992067-A - Method for judging stability of polymerase

CN121992067ACN 121992067 ACN121992067 ACN 121992067ACN-121992067-A

Abstract

The invention discloses a method for judging the stability of polymerase. The invention provides a method for detecting the stability of DNA polymerase, which is to detect the stability of the DNA polymerase by utilizing the principle of reactivating the DNA polymerase after reversible inactivation. The scheme of the invention has simple operation, low requirement on raw materials required by amplification, no need of adding fluorescent components such as radioactive isotopes, fluorescent markers and the like in the amplification process, and no pollution and interference caused by fluorescence. The invention is more suitable for the reaction conditions of reagent development of high-throughput sequencing, whole genome amplification, isothermal amplification, rolling circle replication and other applications, is more suitable for judging the stability of the enzyme, provides reference for judging the use condition of selecting modified enzyme or specific enzyme, and is convenient, time-saving and cost-saving.

Inventors

  • ZHAO WEI
  • GONG MEIHUA
  • XU CHONGJUN

Assignees

  • 深圳华大智造科技股份有限公司

Dates

Publication Date
20260508
Application Date
20241106

Claims (10)

  1. 1. A method for detecting the stability of DNA polymerase is characterized in that the method detects the stability of the DNA polymerase by utilizing the principle of reversibly inactivating and then reactivating the DNA polymerase.
  2. 2. The method according to claim 1, wherein the method comprises the steps of: (A) The first round of amplification, namely catalyzing the polymerization of substrate dNTP molecules to form child DNA by using the parent DNA as a template and using DNA polymerase to be detected; (B) Reversibly inactivating the DNA polymerase to be detected after the first round of amplification, and detecting the amount of the progeny DNA obtained after the first round of amplification; (C) And (3) carrying out renaturation on the DNA polymerase to be detected after the treatment of the step (B) under different test conditions, then carrying out a second round of amplification, detecting the quantity of the progeny DNA obtained after the second round of amplification, and further determining the activity change of the DNA polymerase under different test conditions.
  3. 3. The method according to claim 1 or 2, wherein (C) is carried out as follows: (C1) Test and control groups were set as follows: a test group for treating the DNA polymerase to be tested deactivated in the step (B) under the different test conditions and then renaturating the DNA polymerase to be tested; A control group in which the DNA polymerase to be tested which has been inactivated in said step (B) is directly renatured; (C2) A second round of amplification, namely continuously catalyzing the polymerization of substrate dNTP molecules to form filial generation DNA by utilizing the DNA polymerase to be detected after renaturation in the test group and the control group in the (C1); (C3) And detecting the amounts of the progeny DNA obtained after the second round of amplification in the test group and the control group respectively, and comparing the amounts, so as to determine the activity change of the DNA polymerase under different test conditions.
  4. 4. The method according to claim 2 or 3, wherein the reversible inactivation and the renaturation are performed as follows: (a1) Performing the reversible deactivation using a chelating agent, performing the renaturation using a metal ion; (a2) Carrying out the reversible deactivation under acidic conditions and the renaturation under alkaline conditions; (a3) Performing the reversible inactivation using a denaturing agent and the renaturation using an antioxidant; (a4) The reversible deactivation is performed using an ionic surfactant and the renaturation is performed using a nonionic surfactant.
  5. 5. The method according to claim 4, wherein in (a 1), the chelating agent is EDTA and the metal ion is Mg 2+ , or In (a 2), the acid is a weak acid and the base is a weak base, or In (a 3), the denaturant is urea or guanidine hydrochloride or guanidine isothiocyanate, and the antioxidant is cysteine, ascorbic acid, beta-mercaptoethanol or DTT, or In (a 4), the ionic surfactant is SDS and the nonionic surfactant is Tween20 or NP-40.
  6. 6. The method according to any one of claims 1 to 5, wherein the different test conditions are different temperatures, different humidities, different pH, different concentrations of denaturant and/or different strength of mechanical force; Or (b) In the method, detecting the amount of the progeny DNA is performed according to any one of the following methods: (b1) Incorporating fluorescent molecules into the daughter DNA, and detecting the quantity of the daughter DNA by the intensity of fluorescent signals; (b2) Detecting the amount of said progeny DNA by measuring the light absorbance at 260 nm; (b3) The amount of the progeny DNA was detected by agarose gel electrophoresis.
  7. 7. The method according to claim 1 to 6, wherein the parent DNA is linear single-stranded DNA, circular single-stranded DNA or double-stranded DNA.
  8. 8. The method of claim 1 to 7, wherein the amplification is rolling circle amplification, polymerase chain reaction or multiplex strand displacement amplification.
  9. 9. Use of the method of any one of claims 1-8 in any one of the following: (A1) Screening for DNA polymerase variants; (A2) Determining conditions under which the DNA polymerase is used; (A3) Developing DNA polymerase related reagents; (A4) The activity of the DNA polymerase under different conditions was indirectly determined.
  10. 10. The method of claim 9, wherein the reagent is a high throughput sequencing reagent, a whole genome amplification reagent, a rolling circle amplification reagent, a polymerase chain reaction reagent, or a multiplex strand displacement amplification reagent.

Description

Method for judging stability of polymerase Technical Field The invention relates to the technical field of biology, in particular to a method for judging the stability of polymerase. Background The DNA polymerase, also called DNA-DEPENDENT DNA polymerase (DNA pol), is an enzyme which takes parent DNA as a template and catalyzes the polymerization of substrate dNTP molecules to form progeny DNA, is a very important molecular biological tool enzyme, can be used for whole genome amplification, isothermal amplification, rolling circle replication, DNA sequencing, biochemical analysis, virus detection, nucleic acid detection and the like, and has important roles in the fields of genetic engineering, high-throughput sequencing, molecular diagnosis, medical detection and the like. In order for the modified or unmodified enzyme to exhibit the best activity and achieve the best effect in the above different fields of application, the enzyme used under the conditions of biochemical reaction (e.g., stronger acidity or alkalinity, higher temperature, heavy metals, high salts, denaturants) needs to be tested to determine the stability of the enzyme. While catalytically active enzyme molecules are held by a delicate balance of non-covalent forces (hydrophobic, ionic and van der Waals forces and hydrogen bonding). When the enzyme is exposed to a concentration of denaturing agents or adverse environmental conditions, the enzyme molecule exhibits little or more partial extension, its active sites are damaged, activity is reduced or lost. Therefore, the stability of DNA polymerase is usually determined by measuring the activity of the enzyme after treatment under different conditions. The stability of the DNA polymerase is accurately judged, the optimal effect in different application fields is ensured, and the determination of the enzyme activity is vital. A conventional polymerase assay is described as follows (1) radioisotope labelling, which relies on the incorporation of a radioactive element 3H labelled nucleotide. Namely, the activity of the enzyme was calculated by measuring the amount of radioisotope in the acid-insoluble product by synthesizing a radiolabeled nucleotide chain under the reaction system and conditions of Phi29DNA polymerase. (2) PCR was performed using a primer with cy5 fluorescent label at the 5' end, and the reaction was stopped by adding an equal volume of stop solution containing a competitive oligonucleotide at different times, which prevented recombination of the fluorescent product with the template. The results were then verified by electrophoresis using polyacrylamide containing 8M urea. (3) According to the fluorescent dye doping method, SYBR GreenI is doped in a PCR reaction system, the fluorescence intensity can be gradually increased along with the progress of the reaction, the initial rate of the reaction can be obtained through images given by a real-time fluorescent quantitative PCR instrument, the concentration of added enzyme is different, the initial rate is also different, according to the characteristic, a standard curve can be drawn by using the enzyme with known enzyme activity, and the initial rate of the unknown enzyme reaction is brought into the standard curve, so that the enzyme activity of the unknown enzyme can be obtained. These methods have drawbacks. The radioisotope labeling method is easy to produce radioactive pollution, needs a plurality of steps such as precipitation, filtration and the like, has long period, is difficult to realize high flux and automation, brings difficulty to screening, and has higher cost because fluorescence labeling is needed by a PCR method using a primer with cy5 fluorescence labeling at the 5' end and higher requirement on the specificity of the primer because of a fluorescent dye doping method. Disclosure of Invention The present invention claims a method for determining the stability of a polymerase. In a first aspect, the invention claims a method for detecting the stability of a DNA polymerase. The method for detecting the stability of the DNA polymerase disclosed by the invention is to detect the stability of the DNA polymerase by utilizing the principle that the DNA polymerase is reversibly inactivated and then is activated. The detection of the stability of the DNA polymerase is specifically to determine the activity change of the DNA polymerase under different test conditions. Further, the method may comprise the steps of: (A) The first round of amplification, namely catalyzing the polymerization of substrate dNTP molecules to form child DNA by using the parent DNA as a template and using DNA polymerase to be detected; (B) Reversibly inactivating the DNA polymerase to be detected after the first round of amplification (in an original reaction system), and detecting the amount of the progeny DNA obtained after the first round of amplification; (C) And (3) carrying out renaturation on the DNA polymerase to be detected after the tre