CN116064468B - Chemically modified Taq enzyme storage solution - Google Patents
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- CN116064468B CN116064468B CN202211375240.0A CN202211375240A CN116064468B CN 116064468 B CN116064468 B CN 116064468B CN 202211375240 A CN202211375240 A CN 202211375240A CN 116064468 B CN116064468 B CN 116064468B
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 94
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 94
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- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 4
- 229930195725 Mannitol Natural products 0.000 description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
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- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000012536 storage buffer Substances 0.000 description 3
- -1 DTT Chemical compound 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- ZYFVNVRFVHJEIU-UHFFFAOYSA-N PicoGreen Chemical compound CN(C)CCCN(CCCN(C)C)C1=CC(=CC2=[N+](C3=CC=CC=C3S2)C)C2=CC=CC=C2N1C1=CC=CC=C1 ZYFVNVRFVHJEIU-UHFFFAOYSA-N 0.000 description 2
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 2
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- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- 125000003739 carbamimidoyl group Chemical group C(N)(=N)* 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 2
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
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- 125000000647 trehalose group Chemical group 0.000 description 1
- 238000005583 trifluoroacetylation reaction Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The application provides a storage solution for chemically modifying Taq enzyme and application thereof, belonging to the technical field of biology. The trehalose and/or sorbitol are added into the chemical modification Taq enzyme storage solution, so that the activity of the chemical modification Taq enzyme can be maintained when the temperature is changed, and the subsequent PCR reaction can not be inhibited.
Description
Technical Field
The application relates to the technical field of biology, in particular to a chemical modification Taq enzyme storage solution.
Background
The chemical modification Taq enzyme is characterized in that a chemical group is subjected to chemical reaction with an epsilon-amino group on lysine in the Taq enzyme, so that the epsilon-amino group of the Taq enzyme forms a new chemical bond to be connected with a new chemical group, the change of the chemical group on the lysine can cause the three-dimensional interaction or the configuration change of the protein, and further the inactivation of the protein is caused, and the inactivation can be performed through reversible reaction in the incubation process after the temperature is increased, so that the activity of the protein is recovered. For example, chemical modification Taq enzymes such as citraconylation, tetrafluorosuccinylation, acetoacetylation, maleylation, amidination, trifluoroacetylation, hydroformylation and the like reported in the literature all achieve the hot start effect of the Taq enzyme by the principle. The chemical modification Taq enzyme has the further characteristic that a part of DNA polymerase activity is released along with the PCR process of a high-temperature denaturation step, so that the chemical modification Taq enzyme is subjected to slow release during the PCR cycle, and the chemical modification Taq enzyme is one of reasons for better specificity of the modified enzyme.
As qPCR technology is used in the diagnostic industry, the need for various indicators in the qPCR process is also becoming more stringent. Such as the need for long-term storage stability of the enzyme, or the reduction of enzyme activity loss as much as possible in some pressurized experiments, to avoid variations in the detection index (qPCR detection rate or yield) due to loss of enzyme activity. US6183998B1 discloses a base for a Taq enzyme storage buffer (20 mM Tris-HCl,100mM KCl, 1mM DTT, 0.1mM EDTA, 0.5% Tween 20, 50% Glycerol, 0.5% NP-40), however, there are few reports on storage buffers for chemically modifying Taq enzyme. The application discovers that the basic formula of the Taq enzyme storage solution is used for storing the chemical modification Taq enzyme, and the enzyme activity can be obviously reduced after the basic formula is stored for 3 days at 37 ℃, so that the amplification performance of the chemical modification enzyme is affected.
Disclosure of Invention
The application aims at overcoming the defects of the prior art and provides a storage solution for chemically modified Taq enzyme, which can remarkably improve the storage stability of the chemically modified Taq enzyme.
In a first aspect, the application provides a stock solution of chemically modified Taq enzyme comprising Tris-HCl, KCl, NP-40, tween-20, EDTA, DTT, glycerol and an additive selected from at least one of trehalose, sucrose, glucose, raffinose, sorbitol, mannitol, betaine, PEG2000 and PEG8000, for example at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight and at least nine, preferably one or two.
In some embodiments, the concentration of Tris-HCl in the stock solution is 15-25mM, preferably 18-22mM, most preferably 20mM; KCl is present at a concentration of 50-150mM, preferably 80-120mM, most preferably 100mM; the concentration of NP-40 is 0.2% to 0.8%, preferably 0.4% to 0.6%, most preferably 0.5%; the concentration of Tween-20 is 0.2% to 0.8%, preferably 0.4% to 0.6%, most preferably 0.5%; EDTA is present at a concentration of 0.05-0.2mM, preferably 0.08-0.15mM, most preferably 0.1mM; the concentration of DTT is 0.5-2mM, preferably 0.8-1.5mM, most preferably 1mM; the concentration of glycerol is 20% to 70%, preferably 40% to 60%, most preferably 50%.
In some embodiments, the concentration of glucose is 50-500mM, preferably 100-300mM, including 100mM,150mM,200mM,250mM,300mM within the stated range; the raffinose concentration is 0.5% -10%, preferably 1% -5%, including 1%,2%,3%,4% and 5% within the stated range; the concentration of PEG2000 is 0.5% -10%, preferably 1% -5%, including 1%,2%,3%,4% and 5% of the range; the concentration of PEG8000 is 0.5% -10%, preferably 1% -5%, including 1%,2%,3%,4% and 5% within the stated range.
In some embodiments, the additive is selected from at least one of trehalose, sucrose, sorbitol, mannitol, and betaine, e.g., at least one, at least two, at least three, at least four, and at least five, preferably one or two.
In some embodiments, the additive is trehalose and/or sorbitol.
In some embodiments, the sucrose concentration is 50-500mM, preferably 100-300mM, including 100mM,150mM,200mM,250mM,300mM within the stated range; the mannitol concentration is 50-500mM, preferably 100-300mM, including 100mM,150mM,200mM,250mM,300mM in the range; the betaine concentration is 200-1000mM, preferably 400-800mM, including 400mM,500mM,600mM,700mM,800mM in the range.
In some embodiments, the trehalose is at a concentration of 10-600mM, preferably 20-500mM, more preferably 50-400mM, including 50mM,60mM,70mM,80mM,90mM,100mM,125mM,150mM,175mM,200mM,225mM,250mM,275mM,300mM,325mM,350mM,375mM,400mM, within the ranges described.
In some embodiments, the sorbitol is at a concentration of 10-600mM, preferably 20-500mM, more preferably 50-400mM, including 50mM,60mM,70mM,80mM,90mM,100mM,125mM,150mM,175mM,200mM,225mM,250mM,275mM,300mM,325mM,350mM,375mM,400mM, within the ranges described.
The second aspect of the application provides the use of the stock solution in preserving chemically modified Taq enzyme.
In some embodiments, the chemically modified Taq enzyme is an anhydride modification. In some embodiments, the chemically modified Taq enzyme is a citraconyl modification. In some embodiments, the chemically modified Taq enzyme is a tetrafluorosuccinyl modification. In some embodiments, the chemically modified Taq enzyme is acetyl modified. In some embodiments, the chemically modified Taq enzyme is an amidino modification. In some embodiments, the chemically modified Taq enzyme is a trifluoroacetyl modification. In some embodiments, the chemically modified Taq enzyme is an aldehyde group modification.
In a third aspect the application provides a reagent composition comprising chemically modified Taq enzyme and a stock solution according to the first aspect of the application.
In some embodiments, the chemically modified Taq enzyme is an anhydride modification. In some embodiments, the chemically modified Taq enzyme is a citraconyl modification. In some embodiments, the chemically modified Taq enzyme is a tetrafluorosuccinyl modification. In some embodiments, the chemically modified Taq enzyme is acetyl modified. In some embodiments, the chemically modified Taq enzyme is an amidino modification. In some embodiments, the chemically modified Taq enzyme is a trifluoroacetyl modification. In some embodiments, the chemically modified Taq enzyme is an aldehyde group modification.
In some embodiments, the reagent composition has a percent retention of enzyme activity of greater than 90% after treatment under warming conditions, in some embodiments, 37 ℃ for 3 days.
In a third aspect the application provides an amplification kit comprising a stock solution as provided in the first aspect of the application or a reagent composition as provided in the third aspect.
In some embodiments, the amplification kit further comprises auxiliary detection reagents, such as pure water and dntps.
Advantageous effects
According to the application, aiming at the development research of the storage buffer solution of the chemical modification Taq enzyme, trehalose and sorbitol are added into the basic formula of the Taq enzyme, so that the storage stability of the chemical modification enzyme at 37 ℃ for 3 days can be remarkably improved, the enzyme activity of the chemical modification enzyme is reserved, and the subsequent qPCR amplification performance is not influenced.
Drawings
Fig. 1: the three chemically modified Taq enzymes are respectively placed at-20 ℃ and 37 ℃ for 3 days and then subjected to PCR amplification;
fig. 2: influence of different stock solutions on the PCR amplification performance of the AceTaq enzyme;
fig. 3: influence of different stock solutions on the RT-PCR amplification performance of AceTaq enzyme.
Detailed Description
The technical scheme of the application is further described below by means of specific embodiments in combination with the accompanying drawings. However, the following examples are merely illustrative of the present application and are not representative or limiting of the scope of the present application. The protection scope of the application is subject to the claims. In the examples below, reagents and consumables were purchased from commercial suppliers, and experimental methods and techniques were used as conventional in the art, unless otherwise specified.
Example 1
Three types of chemically modified Taq enzymes from different manufacturers: 5U/. Mu.l AceTaq (Vazyme, P401, anhydride modification), 5U/. Mu.l FastStart Taq (Roche, 12032937001, anhydride modification) and 5U/. Mu.l AmpliTaq (ABI, N808-0241, formaldehyde modification) were the subjects, and they were divided into two groups, wherein the untreated group was placed at-20℃for 3d, and the treated group was placed at 37℃for 3d.
Enzyme activity was measured for the treated and untreated groups of enzymes, respectively: 1. and (3) standard curve preparation: taq enzyme labels (Vazyme, P101) were subjected to gradient dilution to 15 mU/. Mu.l, 7.5 mU/. Mu.l, 3.75 mU/. Mu.l, 1.875 mU/. Mu.l, 0.9375 mU/. Mu.l, 0 mU/. Mu.l, 50. Mu.l each were taken with M13 Mix (M13 single-stranded template 50ng, single-stranded oligonucleotide 0.1. Mu.M, dNTP 0.2mM,Tris HCl 10mM,KCl 50mM,MgCl) 2 1.5mM, pH 8.0). PCR was performed at 72℃for 30min. And (3) adding a certain amount of PicoGreen into the reaction product, reading the fluorescence value by an enzyme-labeled instrument, and drawing an enzyme activity standard curve according to the fluorescence value of each concentration. 2. And (3) measuring the enzyme activity of a sample to be measured: gradient dilution of the chemically modified Taq enzyme to be tested to 6 mU/. Mu.l and 3 mU/. Mu.l, 50. Mu.l each with M13 Mix (M13 single-stranded template 50ng, single-stranded oligonucleotide 0.1. Mu.M, dNTP 0.2mM,Tris HCl 10mM,KCl 50mM,MgCl) 2 1.5mM, pH 8.0), and PCR was performed at 72℃for 30min. And adding a certain amount of PicoGreen into the reaction product, reading a fluorescence value by an enzyme-labeled instrument, substituting the measured fluorescence value into a standard Taq enzyme fluorescence curve to convert the enzyme activity of the to-be-detected product, and taking the average value of the two as a final result. The results are shown in Table 1, in which the enzyme activity retention after pressurization was significantly reduced.
TABLE 1 Activity of three chemically modified Taq enzymes after 3 days treatment at 37℃and Activity residual Rate
| Chemically modified enzymes | -20℃ | 37 ℃ for 3 days | Residual percentage of activity |
| AceTaq | 5.33U/μl | 3.62U/μl | 68％ |
| FastStart Taq | 5.1U/μl | 3.16U/μl | 62％ |
| AmpliTaq | 5.46U/μl | 3.86U/μl | 76％ |
Example 2
The PCR amplification experiments were performed using the 293 genome as a template, using the treated and untreated group enzymes of example 1, respectively, and the PCR systems and procedures were as follows.
The length of the amplified product was 1800bp, and the used upstream and downstream primers (direction: 5 '. Fwdarw.3') were:
upstream primer F (SEQ ID NO. 1): TTACTCACCCGACATCTCCTC
Downstream primer R (SEQ ID NO. 2): GCAGCAGGAAAGCTACCGA
TABLE 2 PCR System
| 10xPC Buffer (without dNTP) a | 2.5μl |
| dNTP Mix(10mM each) b | 0.5μl |
| 5xPCR Enhancer c | 5μl |
| Taq DNA Polymerase(5U/μl) | 0.5μl |
| Upstream primer/downstream primer | 0.5/0.5μl |
| Template DNA (10 ng/. Mu.l) | 2μl |
| Pure water | Supplement to 25. Mu.l |
a：10xPCR Buffer(Vazyme，P402)；b、dNTP Mix(Vazyme，P402)；c、5x PCR Enhancer(Vazyme，P402)；
TABLE 3 PCR procedure
The PCR amplification experimental results are shown in FIG. 1, wherein the PCR yield after 3d post-treatment of AceTaq and FastStartTaq chemical modification enzymes at 37 ℃ is obviously reduced.
Example 3
With stock solution 1 (20 mM Tris-HCl,100mM KCl,0.5%NP-40 (volume ratio), 0.5% Tween-20 (volume ratio), 0.1mM EDTA,1mM DTT,50% glycerol (volume ratio)) as a control, stock solutions 2-34 were prepared by adding 11 additives, respectively, wherein the kinds and the content of each additive are shown in Table 4.
TABLE 4 Table 4
5U of AceTaq (Vazyme, P401) was added to each of the stock solutions 1 to 34, and divided into two groups, one group was left at-20℃for 3 days and the other group was left at 37℃for 3 days. The method for measuring the enzyme activity was the same as in example 1, and the results of screening the additives based on the percentage of enzyme retention activity are shown in Table 4.
TABLE 5 influence of different additives on the enzyme Activity after 3 days at 37℃after addition (unit: U/. Mu.l)
The results show that the addition of trehalose, sorbitol, betaine, mannitol, sucrose and polyethylene glycol with a certain concentration in the storage solution 1 can obviously improve the retention activity of the AceTaq enzyme after 3d at 37 ℃, wherein the enzyme retention activity of 200mM trehalose is obviously improved by 22% compared with the control group, and the enzyme retention activity of 200mM sorbitol is obviously improved by 23% compared with the control group.
Further, 200mM trehalose and 200mM sorbitol were added simultaneously to the stock solution 1 to prepare a stock solution 35. The results of the enzyme treatment and enzyme retention activity measurement are shown in Table 6, which were the same as those in the foregoing example 2. Trehalose and sorbitol are added into the storage solution at the same time, and after the storage solution is treated for 3 days at 37 ℃, the enzyme activity retention is improved by 26% compared with a control group.
TABLE 6 influence of enzyme Activity after 3 days at 37℃after addition of trehalose and sorbitol (unit: U/. Mu.l)
| Storage liquid | -20℃ | 37℃，3d | Percentage of enzyme retained activity |
| 1 | 5.5 | 3.91 | 71％ |
| 35 | 5.3 | 5.13 | 97％ |
Example 4
To further investigate the effect of trehalose and/or sorbitol addition to the storage buffer on the amplification performance of chemically modified Taq enzyme 2.5U of AceTaq enzyme (Vazyme, P401) was kept in storage solutions 1, 15, 18 and 35 respectively and divided into a treatment group and a control group, wherein the control group was left at-20 ℃ for 3d and the treatment group was left at 37 ℃ for 3d. Subsequently, a PCR experiment was performed under the same system using 293 genome as a template to examine its effect on PCR amplification yield, wherein the PCR reaction system and procedure were the same as in example 2.
As shown in fig. 2, the amplification yields of the PCR reactions using AceTaq enzymes in stock solutions 15, 18 and 35 were not significantly different from that of stock solution 1 at-20 ℃, whereas the amplification yields of the PCR reactions using AceTaq enzymes in stock solutions 15, 18 and 35 were significantly higher than that of stock solution 1 after 37 ℃ treatment, indicating that the addition of trehalose and sorbitol to the stock solutions significantly improved the storage stability of AceTaq enzymes at 37 ℃ and did not affect the PCR amplification performance.
Example 5
To further verify whether the addition of trehalose and/or sorbitol to the chemically modified Taq enzyme stock solution affected the subsequent PCR reactions, RT-PCR experiments were performed using 293 genome as a template and the chemically modified Taq enzyme AceTaq (Vazyme, P401) stored in stock solutions 15, 18 and 35, respectively, under the same system, the RT-PCR reaction system and procedure were as shown in tables 6 and 7, and the upstream and downstream primer and probe sequences (direction: 5 '. Fwdarw.3') were as shown in Table 8, respectively.
TABLE 6 RT-PCR reaction System
a：10xPCR Buffer(Vazyme，P402)；b、dNTP Mix(Vazyme，P402)；
TABLE 7 RT-PCR procedure
TABLE 8 RT PCR primer probe sequences
As shown in FIG. 3, the amplification curves for stock 15, 18 and 35 had no significant effect on the sensitivity and plateau of RT-PCR compared to stock 1. Therefore, it was demonstrated that the addition of trehalose and/or sorbitol to the chemically modified Taq enzyme stock solution did not inhibit the PCR reaction.
Claims (9)
1. A chemically modified Taq enzyme stock solution comprising 18-22mM Tris-HCl, 80-120mM KCl, 0.4% -0.6% NP-40, 0.4% -0.6% Tween-20, 0.08-0.15mM EDTA, 0.8-1.5mM DTT, 40% -60% glycerol and an additive selected from one or both of 100-300mM trehalose and 100-300mM sorbitol.
2. The stock solution of claim 1, wherein Tris-HCl has a concentration of 20mM, KCl has a concentration of 100mM, NP-40 has a concentration of 0.5%, tween-20 has a concentration of 0.5%, EDTA has a concentration of 0.1mM, DTT has a concentration of 1mM, and glycerol has a concentration of 50%.
3. The stock solution of claim 1, wherein the trehalose is at a concentration of 200mM.
4. The stock solution of claim 1, wherein the sorbitol is at a concentration of 200mM.
5. Use of a stock solution according to any one of claims 1-4 for preserving chemically modified Taq enzyme.
6. The use of claim 5, wherein the chemically modified Taq enzyme is an anhydride modified Taq enzyme.
7. A reagent composition comprising the stock solution of any one of claims 1-4 and a chemically modified Taq enzyme.
8. The reagent composition of claim 7, wherein the chemically modified Taq enzyme is an anhydride modified Taq enzyme.
9. An amplification kit comprising the reagent composition of claim 7.
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| CN1940087A (en) * | 2006-08-18 | 2007-04-04 | 上海科华生物工程股份有限公司 | Method for inspecting hepatitis and AIDS virus nucleic acid by synchronous amplification and its reagent kit |
| CN112831551A (en) * | 2021-01-22 | 2021-05-25 | 武汉明德生物科技股份有限公司 | Fully-premixed RT-PCR reaction system and application thereof |
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| EP3645710A1 (en) * | 2017-06-26 | 2020-05-06 | Thermo Fisher Scientific Baltics Uab | Thermophilic dna polymerase mutants |
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| CN1940087A (en) * | 2006-08-18 | 2007-04-04 | 上海科华生物工程股份有限公司 | Method for inspecting hepatitis and AIDS virus nucleic acid by synchronous amplification and its reagent kit |
| CN112831551A (en) * | 2021-01-22 | 2021-05-25 | 武汉明德生物科技股份有限公司 | Fully-premixed RT-PCR reaction system and application thereof |
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