Enzyme additive with enhancing and protecting effects and application thereof
阅读说明：本技术 一种增强和保护作用的酶添加剂及其应用 (Enzyme additive with enhancing and protecting effects and application thereof ) 是由 吴诗扬 许嘉森 彭璨璨 刘志明 于 2019-11-14 设计创作，主要内容包括：本发明提供了一种增强和保护作用的酶添加剂,其包括增强组分和保护组分两部分,所述增强组分包括有：1.8-2.2M甜菜碱、0.6-1％BSA、90-110mM PVP、0.02-0.04mM DTT、90-110mM环糊精、5-7％Triton X-100；所述保护组分包括有：40-45％甘油、0.3-0.5％黄原胶、2.2-2.6％海藻酸钠、5-7％PEG-400、1.2-1.5％硫酸铵、1-1.4％柠檬酸钾。本发明还提供了添加上述酶添加剂的扩增体系。本发明经过合理配制的酶添加剂的组分可以增加聚合酶的热稳定性及其活性,增强DNA聚合酶、引物和模板的结合,促进PCR反应起始复合物形成,增加PCR特异性扩增,提高扩增效率。(The invention provides an enzyme additive with enhancing and protecting functions, which comprises two parts of an enhancing component and a protecting component, wherein the enhancing component comprises: 1.8-2.2M betaine, 0.6-1% BSA, 90-110mM PVP, 0.02-0.04mM DTT, 90-110mM cyclodextrin, 5-7% Triton X-100; the protective component comprises: 40-45% of glycerol, 0.3-0.5% of xanthan gum, 2.2-2.6% of sodium alginate, 5-7% of PEG-400, 1.2-1.5% of ammonium sulfate and 1-1.4% of potassium citrate. The invention also provides an amplification system added with the enzyme additive. The components of the enzyme additive which are reasonably prepared can increase the thermal stability and the activity of polymerase, enhance the combination of DNA polymerase, primers and templates, promote the formation of PCR reaction initial complexes, increase the PCR specific amplification and improve the amplification efficiency.)
1. An enzyme additive for enhancing and protecting action, which is characterized by comprising two parts of an enhancing component and a protecting component, wherein,
the reinforcing component comprises: 1.8-2.2M betaine, 0.6-1% BSA, 90-110mM PVP, 0.02-0.04mM DTT, 90-110mM cyclodextrin, and 5-7% Triton X-100;
the protective component comprises: 40-45% of glycerol, 0.3-0.5% of xanthan gum, 2.2-2.6% of sodium alginate, 5-7% of PEG-400, 1.2-1.5% of ammonium sulfate and 1-1.4% of potassium citrate.
2. The enhanced-and-protected enzyme additive according to claim 1, wherein said enhancing component comprises: 1.9-2.1M betaine, 0.7-0.9% BSA, 95-105mM PVP, 0.025-0.035mM DTT, 95-105mM cyclodextrin, and 5.5-6.5% Triton X-100;
the protective component comprises: 41-43% of glycerol, 0.35-0.45% of xanthan gum, 2.3-2.5% of sodium alginate, 5.5-6.5% of PEG-400, 1.35-1.45% of ammonium sulfate and 1.1-1.3% of potassium citrate.
3. The enhanced-and-protected enzyme additive according to claim 2, wherein said enhancing component comprises: 2M betaine, 0.8% BSA, 100mM PVP, 0.03mM DTT, 100mM cyclodextrin, and 6% Triton X-100;
the protective component comprises: 42% glycerol, 0.4% xanthan gum, 2.4% sodium alginate, 6% PEG-400, 1.4% ammonium sulfate, and 1.2% potassium citrate.
4. The enhanced-and-protected enzyme additive according to any one of claims 1-3, wherein the volume ratio of said protecting component to said enhancing component is 1: 9-11.
5. The enhanced-and-protected enzyme additive according to claim 4, wherein the volume ratio of said protecting component to said enhancing component is 1: 10.
6. use of the enhanced and protected enzyme additive of any one of claims 1-5 in polymerase chain reaction.
7. Polymerase chain reaction system, characterized in that it contains an enzyme additive according to any one of claims 1 to 5.
8. The polymerase chain reaction system of claim 7 wherein the volume ratio of the enhancing component to the protecting component is 1: 9-11.
9. The polymerase chain reaction system of claim 8, wherein the volume ratio of the enhancing component to the protecting component is 1: 10.
10. the polymerase chain reaction system of any of claims 7-9, wherein the polymerase chain reaction is any of RT-PCR, immuno-PCR, nested PCR, fluorescence PCR, in situ PCR, membrane-bound PCR, anchored PCR, anchorage PCR, in situ PCR, asymmetric PCR, long-range PCR, parachute PCR, gradient PCR.
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to an enzyme additive with enhancing and protecting effects and application thereof.
Background
Nucleic acid detection covers many fields such as gene screening, biomedical research and the like, and is closely related to social stability and human health. How to overcome the defects of complex operation, time and labor waste, low sensitivity and the like of the traditional nucleic acid detection method, and developing and perfecting a high-sensitivity, high-accuracy, rapid, simple and convenient research and analysis method is a hotspot problem for researches of researchers for many years.
The nucleic acid detection has very important significance in the life science research, the signal amplification detection of the nucleic acid is an important detection means in the nucleic acid molecule detection, the signal method detection technology of the nucleic acid molecule selectively combines various tool enzymes and nanoparticles with electrochemical, optical and other technologies, so that the high-sensitivity and high-specificity detection of target molecules is realized, and the detection method has the advantages of quick reaction, good selectivity, high sensitivity, no need of particularly expensive instruments and equipment and the like, has great application potential in the nucleic acid detection, is a new technology and a new method in the life science research, and quickly becomes a research hotspot and a research focus in the biochemical analysis field. How to fully utilize various biological and biochemical methods to realize high-sensitivity and high-selectivity detection of nucleic acid in a biological system is a new problem in life science research and biochemical analysis. The problems of further improving the detection sensitivity, reducing the interference of complex environment and the like are still the current research hotspots and difficulties.
The tool enzyme has excellent characteristics of high specific sequence recognition capability, high catalytic activity and the like, and can perform a series of connection, cutting or modification on a DNA sequence under a certain condition, so that the functions of the tool enzymes are very important.
One of the commonly used tool enzymes, Taq DNA polymerase, is widely applied to the fields of medicine, chemical industry, food, clinical and chemical analysis and the like as a biocatalyst with excellent performances such as high substrate specificity, high catalytic efficiency and the like. However, the common liquid enzyme preparation is inconvenient to carry, transport and store due to large volume, and in order to expand the application range of the enzyme, the optimization aiming at the enzyme preparation product at present mainly has the following 4 aspects: study of enzymes in thermophilic organisms; studies on protein denaturation; chemical modification of protein molecules; auxiliary enhancement effect of the additive on the enzyme.
The first three aspects are mainly to optimize and improve the enzyme by performing related editing or modification from the gene or protein level, the research period is long, and the practical implementation difficulty is high, so that the research and discovery of an enzyme additive or an additive combination are one of the most practical optimization methods in comprehensive consideration at present, the common additive can partially improve the corresponding performance, but when the amplification reaction is performed, a large fragment amplification product of a non-target can be generated due to the repeated sequence existing at the position of a part of primers, the large fragment amplification product is caused by the existence of a plurality of primer binding sites in the template DNA, is specifically amplified, and is a non-target product to be removed, and under the condition, the ideal optimization effect cannot be achieved by the common PCR additive. In view of this, the present invention is proposed.
Disclosure of Invention
Based on this, it is an object of the present invention to provide an enhanced and protective enzyme additive which has a significant fluorescence enhancement effect and can improve the sensitivity and specificity of detection.
The technical scheme for realizing the purpose is as follows:
an enhancing and protecting enzyme additive, which comprises an enhancing component and a protecting component,
the reinforcing component: 1.8-2.2M betaine, 0.6-1% BSA, 90-110mM PVP, 0.02-0.04mM DTT, 90-110mM cyclodextrin, and 5-7% Triton X-100;
a protective component: 40-45% of glycerol, 0.3-0.5% of xanthan gum, 2.2-2.6% of sodium alginate, 5-7% of PEG-400, 1.2-1.5% of ammonium sulfate and 1-1.4% of potassium citrate.
In some of these embodiments, the reinforcing component: 1.9-2.1M betaine, 0.7-0.9% BSA, 95-105mM PVP, 0.025-0.035mM DTT, 95-105mM cyclodextrin, and 5.5-6.5% Triton X-100;
a protective component: 41-43% of glycerol, 0.35-0.45% of xanthan gum, 2.3-2.5% of sodium alginate, 5.5-6.5% of PEG-400, 1.35-1.45% of ammonium sulfate and 1.1-1.3% of potassium citrate.
In some of these embodiments, the reinforcing component: 2M betaine, 0.8% BSA, 100mM PVP, 0.03mM DTT, 100mM cyclodextrin, 6% Triton X-100;
a protective component: 42% of glycerol, 0.4% of xanthan gum, 2.4% of sodium alginate, 6% of PEG-400, 1.4% of ammonium sulfate and 1.2% of potassium citrate.
In some of these embodiments, the volume usage ratio of the protective component to the reinforcing component is 1: 9-11. More preferably, the volume dosage ratio is 1: 10.
another aspect of the present invention is to provide the use of the above-described enhancing and protecting enzyme additive in polymerase chain reactions.
Another aspect of the present invention is to provide a polymerase chain reaction system containing the above enzyme additive.
In some embodiments, the volume ratio of the enhancing component to the protecting component in the polymerase chain reaction system containing the enzyme additive is 1: 9-11. More preferably, the volume dosage ratio is 1: 10.
the main advantages of the invention are:
1. the reasonably prepared components of the enzyme additive can increase the thermal stability and activity of polymerase, enhance the combination of DNA polymerase, primers and templates, promote the formation of PCR reaction initial complex, increase PCR specific amplification and improve amplification efficiency; meanwhile, the adsorption of enzyme reagents on the tube wall can be reduced (the xanthan gum has a three-stage structure, so that the xanthan gum has good water flow control property, good thickening property, particularly high viscosity under low mass concentration, and hydrophilic and lipophilic groups, so that after being dissolved in water, the insolubility of oil and water phases is weakened, a stable oil-water dynamic balance system can be formed, and the xanthan gum has good suspension property and emulsifying property). In addition, the protective component of the enzyme additive maintains the molecular conformation of an enzyme active center unchanged through acting forces such as hydrogen bonds, static electricity, hydrophobicity and the like, and through modes such as increasing steric hindrance, improving system viscosity, improving the glass transition temperature of enzyme and the like, so that the stability of an enzyme solution system is ensured, and meanwhile, the enzyme additive can be directly combined with PCR inhibitors such as hemoglobin and melanin which possibly exist in the system, so that the influence of the inhibitors on DNA polymerase and the enzyme protective component is prevented.
2. The enzyme additive provided by the invention has flexibility and wide application range, can be directly used for a synergistic reaction aiming at different commercial PCR kits in the market, specifically can be directly added with common commercial Taq enzyme reagents for premixing, can be proportionally added and uniformly mixed when a basic reaction system (namely, amplification necessary components such as Taq DNA polymerase, dNTPs, buffer solution and the like except for a template and a primer) is prepared, and can also be directly used on a machine for detection after the final addition and uniform mixing of the system is completed, so that a detector can adopt various methods to add the enzyme additive to effectively improve the reaction efficiency when in actual use.
3. The enzyme additive provided by the invention can effectively improve the amplification efficiency of the conventional nucleic acid template and obviously enhance the sensitivity of PCR amplification.
Drawings
FIG. 1 is a graph showing a comparison of the results of measurement of
FIG. 2 is a graph showing the results of the test in
fig. 3 is a graph showing the test results of
fig. 4 is a graph showing the test results of the
FIG. 5 is a graph comparing the results of the test of the mutant homozygous sample of example 5, sample No. 44-CYP2C19 x 2G681A, in which the reaction system to which the enhancer was added, Curve 1: FAM, curve 4: VIC; the detection result of the reaction system with the addition of the common PCR additive, curve 3: FAM, curve 2: VIC; test results for the reaction system without additive, curve 6: FAM, curve 5: and (5) VIC.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It will be appreciated that the experimental procedures for the following examples, where specific conditions are not indicated, are generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various reagents used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The enhancing part of the enzyme additive can obviously improve the 5 '-3' exonuclease activity of DNA polymerase, promote the combination of MGB and DNA minor groove and simultaneously have very obvious fluorescence enhancing effect; the protective component of the enzyme additive can effectively prolong and stabilize the activity of the DNA polymerase enhancing component, and simultaneously improve the sensitivity and specificity of detection.
The Polymerase Chain Reaction (PCR) is a molecular biology technique for amplifying and amplifying specific DNA fragments, and can be regarded as special DNA replication in vitro, and the biggest characteristic of the PCR is that trace amount of DNA can be greatly increased. The denaturation and renaturation of DNA are controlled by temperature change, and the in vitro replication of specific gene can be completed by adding designed primer, DNA polymerase and dNTP. The classification includes, but is not limited to, RT-PCR, Immuno Polymerase Chain Reaction (IPCR), nested PCR, fluorescent PCR, in situ PCR, membrane-bound PCR, anchored PCR, in situ PCR, asymmetric PCR, long-range PCR, parachute PCR, gradient PCR.