CN116926160A - Enzyme activity detection kit for T4 DNA ligase and enzyme activity determination method - Google Patents
Enzyme activity detection kit for T4 DNA ligase and enzyme activity determination method Download PDFInfo
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- 102000012410 DNA Ligases Human genes 0.000 title claims abstract description 113
- 108010061982 DNA Ligases Proteins 0.000 title claims abstract description 113
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 33
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 33
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- 238000007169 ligase reaction Methods 0.000 claims abstract description 24
- 108020004682 Single-Stranded DNA Proteins 0.000 claims abstract description 19
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- 239000012634 fragment Substances 0.000 claims description 26
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- 238000000137 annealing Methods 0.000 claims description 19
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 14
- 239000003638 chemical reducing agent Substances 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 12
- 125000003729 nucleotide group Chemical group 0.000 claims description 12
- 239000006174 pH buffer Substances 0.000 claims description 12
- 238000005251 capillar electrophoresis Methods 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 11
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 7
- 239000008118 PEG 6000 Substances 0.000 claims description 7
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 7
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 7
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 7
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 7
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 6
- 239000002738 chelating agent Substances 0.000 claims description 6
- 239000003599 detergent Substances 0.000 claims description 6
- 239000003248 enzyme activator Substances 0.000 claims description 6
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 208000034953 Twin anemia-polycythemia sequence Diseases 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000012141 concentrate Substances 0.000 claims description 4
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- 238000006555 catalytic reaction Methods 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 230000002255 enzymatic effect Effects 0.000 claims description 3
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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 claims description 2
- 229930195725 Mannitol Natural products 0.000 claims description 2
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 claims description 2
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- -1 osmolytes Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 239000001488 sodium phosphate Substances 0.000 claims description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
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- 102000039446 nucleic acids Human genes 0.000 description 3
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
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Classifications
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- 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
- C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/9015—Ligases (6)
-
- 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 invention relates to the field of gene sequencing, in particular to an enzyme activity detection kit for T4 DNA ligase and an enzyme activity detection method. The invention relates to an enzyme activity detection kit for T4 DNA ligase, which comprises a reaction substrate raw material for detecting the activity of the T4 DNA ligase, and also comprises a T4 DNA ligase diluent and a T4 DNA ligase reaction buffer solution. The method solves the defects of complicated operation and complicated steps of the existing activity measurement method; the T4 DNA ligase detection kit and the method for detecting the activity of the T4 DNA ligase provided by the invention have the advantages that the phage single-stranded DNA is not required to be used as a reaction substrate, so that the risk of phage residues is not generated, and a fluorescent group or dye is not required to be used, so that the need of specially preparing a fluorescent enzyme-labeled instrument or a fluorescent spectrophotometer is avoided.
Description
Technical Field
The invention relates to the field of gene sequencing, in particular to an enzyme activity detection kit for T4 DNA ligase and an enzyme activity detection method.
Background
With the assistance of ATP energization, T4 DNA ligase catalyzes the formation of a phosphodiester linkage from the 3 'hydroxyl and 5' phosphate ends on cohesive or blunt-ended double-stranded DNA.
Currently, the amount of enzyme required for experiments is gradually increased with the development of genetic sequencing technology, which is one of widely used biomedical detection means. In the existing gene sequencing technology, a plurality of steps use T4 DNA ligase, so that whether the enzyme activity of the T4 DNA ligase is accurately measured is a crucial factor for influencing the smoothness of the gene sequencing reaction.
In the present stage, in the method for semi-quantitatively measuring the enzyme activity of the T4 DNA ligase, gray analysis is carried out by imaging after gel electrophoresis, so that the defects of influence of acceptation and inaccurate gray analysis exist, the enzyme activity error of the T4 DNA ligase measured by the method is larger, and the resolution ratio of the T4 DNA ligase with similar enzyme activities in different batches is not high. In the present stage, most of the methods for determining the activity of the T4 DNA ligase capable of being precisely quantified use fluorescent signals of fluorescent groups (such as Chinese patent CN 113061645A) or dyes (such as Chinese patent CN 104278090A) as an original data reading mode, particularly a mode of using fluorescent signal changes of the fluorescent groups as a result output is adopted, the cost is high, and because of the existence of a signal reading threshold value of an experimental instrument, the fluorescent signals cannot be too low, so that the use amount of the fluorescent groups or the dyes is large (such as Chinese patent CN 108220386A), and the detection cost is further improved. And most of the substrates are phage single-stranded DNA or synthesized into longer fragments, so that the obtaining cost of phage single-stranded DNA is high. And for use environments where microbial fermentation is required, there is a potential risk of phage residues using phage single stranded DNA, resulting in a disrupted microbial fermentation environment. In the existing method, phage single-stranded DNA is not used as a reaction substrate, and long fragments are synthesized, so that the synthesis cost is high due to the fact that the fragments are long, meanwhile, the types of different synthesized fragments are more, the synthesis cost is further increased, and the detection cost is further increased (like Chinese patent CN 113061645A). The reagent cost of the activity detection method is one of factors which make the measurement threshold of the activity of the T4 DNA ligase higher, and the other factor is the measuring instrument. Most of the existing measurement methods use fluorescence as measurement basis, and the fluorescent signals of the fluorescent groups or dyes are read basically by using a fluorescence microplate reader (such as Chinese patent CN 104278090A) or a fluorescence spectrophotometer (such as Chinese patent CN 113061645A), so that for some users, the fluorescence microplate reader or the fluorescence spectrophotometer needs to be specially prepared, and the generated hardware cost further increases the enzyme activity measurement threshold of the T4 DNA ligase. On the other hand, because the T4 DNA ligase is an enzyme which can catalyze the reaction at normal temperature or low temperature, the more and more complicated operation steps are, the greater the possibility of generating operation errors is, the lower the fault tolerance, the further the threshold for implementing the operation is improved, and the practical feasibility and the operation stability are reduced.
The method for measuring the activity of the T4 DNA ligase has the defects of complex operation and complex steps, and has high substrate fragment synthesis cost and high experimental instrument cost on the realization cost, so that the valve threshold for measuring is higher. The complex test conditions and longer time are also one of the reasons for inconvenient test of T4 DNA ligase.
Therefore, the problem of how to rapidly and simply determine the enzyme activity of T4 DNA ligase at low cost is needed to be solved.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to provide an enzyme activity detection kit and an enzyme activity detection method for T4 DNA ligase, which can rapidly and accurately detect the enzyme activity of the T4 DNA ligase, do not need to use phage single-stranded DNA as a reaction substrate or use fluorophores or dyes, and avoid the need of specially preparing a fluorescence microplate reader or a fluorescence spectrophotometer.
The technical scheme for solving the technical problems is as follows:
in a first aspect of the invention, an enzyme activity detection kit for a T4 DNA ligase is provided.
The kit comprises a reaction substrate raw material for detecting the activity of T4 DNA ligase;
the reaction substrate raw material comprises four independent single-stranded DNA (deoxyribonucleic acid) which are divided into two groups;
first strip in first group: 5 'gacctctttatgatggaaatctagt'; the nucleotide sequence numbers are SEQ ID NO:1, a step of;
second strip in the first group: 5 'gtcaactagatttccatcataaagaggtc', nucleotide sequence number SEQ ID NO:2, wherein the second 5' end modifies a phosphate group;
first strip in second group:
5'agatacaagggtccagcaatttctcgtgcgggtgctcaactcaac 3'; the nucleotide sequence numbers are SEQ ID NO:3, a step of;
second strips in the second group:
5'tgacgttgagttgagcacccgcacgagaaattgctggacccttgtatct 3', nucleotide sequence number SEQ ID NO:4, wherein the second 5' end modifies a phosphate group.
Further, the kit also comprises a T4 DNA ligase diluent;
the T4 DNA ligase diluent comprises the following components: pH buffers, chelating agents, detergents, osmolytes, anti-freeze agents and reducing agents.
Preferably, the pH of the T4 DNA ligase dilution is 7.5-8.0.
Preferably, the pH buffer is selected from any one of Tris-HCl, PBS, TAPS, preferably Tris-HCl.
Preferably, the chelating agent is selected from any one of EDTA-2Na, sodium phosphate and DTPA, preferably EDTA-2Na.
Preferably, the detergent is selected from Tween-20, brij 58, triton X-100 or a combination thereof, preferably Tween-20.
Preferably, the osmolality regulator is selected from KCl and CaCl 2 、NaCl、MgCl 2 Or a combination thereof, preferably KCl.
Preferably, the antifreeze is selected from glycerol, ethylene glycol or a combination thereof, preferably glycerol.
Preferably, the reducing agent is selected from DTT, beta-mercaptoethanol, or a combination thereof, preferably DTT.
In a preferred embodiment of the present invention,
the pH buffer is Tris-HCl, and the content of Tris-HCl is 10 mM-60 mM, preferably 15 mM-40 mM, more preferably 20 mM-30 mM, based on the total amount of the T4 DNA ligase diluent.
The chelating agent is EDTA-2Na, and the content of EDTA-2Na is 0.01 mM-10 mM, preferably 0.1 mM-1 mM, based on the total amount of the T4 DNA ligase diluent.
The detergent is Tween-20, and the content of Tween-20 is 0.1% -2% (v/v), preferably 0.5% -1.5% (v/v) based on the total amount of the T4 DNA ligase diluent.
The osmotic pressure regulator is KCl, and the content of KCl is 30 mM-80 mM, preferably 40 mM-60 mM, based on the total amount of the T4 DNA ligase diluent.
The antifreeze is glycerol, and the content of the glycerol is 30% -70% (v/v), preferably 40% -60% (v/v) based on the total amount of the T4 DNA ligase diluent.
The reducing agent is DTT, and the content of the DTT is 0.1 mM-2 mM, preferably 0.5 mM-1.3 mM, based on the total amount of the T4 DNA ligase diluent.
Further, the kit also comprises a T4 DNA ligase reaction buffer solution;
the ligase reaction buffer solution is 10 xT 4 DNA ligase reaction buffer solution and comprises the following components: pH buffers, concentrates, enzyme activators, reducing agents and ATP.
Preferably, the pH of the 10×T4 DNA ligase reaction buffer is 7.5-8.0.
Preferably, the pH buffer is selected from Tris-HCl, PBS or TAPS, preferably Tris-HCl.
Preferably, the concentrate is selected from PEG 6000, mannitol or a combination thereof, preferably PEG 6000.
Preferably, the enzyme activator is selected from MgCl 2 、MgSO 4 Or a combination thereof, preferably MgCl 2 。
Preferably, the reducing agent is selected from DTT, beta-mercaptoethanol, or a combination thereof, preferably DTT.
In a preferred embodiment of the present invention,
the pH buffer is Tris-HCl, and the content of the Tris-HCl is 300 mM-700 mM, preferably 400 mM-500 mM, based on the total amount of 10 xT 4 DNA ligase reaction buffer.
The concentration agent is PEG 6000, and the content of the PEG 6000 is 4% -10% (v/v), preferably 6% -8% (v/v) based on the total amount of the 10 xT 4 DNA ligase reaction buffer solution.
The enzyme activator is MgCl 2 MgCl based on the total amount of 10×T4 DNA ligase reaction buffer 2 The content of (C) is 30 mM-120 mM, preferably 80 mM-100 mM.
The reducing agent is DTT, and the content of DTT is 1 mM-20 mM, preferably 5 mM-13 mM, based on the total amount of 10 xT 4 DNA ligase reaction buffer.
The ATP is contained in an amount of 8 mM-20 mM, preferably 9 mM-11 mM, based on the total amount of the 10 xT 4 DNA ligase reaction buffer.
In a second aspect of the invention, there is provided a method of determining the enzymatic activity of a T4 DNA ligase using a kit as described in the first aspect.
The enzyme activity determination method comprises the following steps:
mixing the two single-stranded DNA fragments of the first group according to the molar ratio of 1:1 at the concentration of 100 mu M, mixing the two single-stranded DNA fragments of the second group according to the molar ratio of 1:1 at the concentration of 100 mu M, and respectively carrying out annealing combination in a common PCR instrument; the annealing process is a slow annealing process, the temperature is slowly reduced from 95 ℃ to 45 ℃ and 1 ℃ is reduced every 10 seconds, so that the two pairs of single strands are completely annealed and combined into two double strands, and the annealing program conditions are set in a common PCR instrument and are shown in the following table:
；
respectively forming two double-stranded DNA containing cohesive ends after annealing and combining, and diluting the products after annealing and combining by 50 times to 1 mu M;
2) Diluting the T4 DNA ligase to be detected by 1000 times by using a T4 DNA ligase diluent, wherein a detection reaction system is configured as follows, and each 20 mu L of the detection reaction system contains 5 mu L of a first group of annealing binding products; 5 μl of the second set of annealed binding products; 0.4. Mu.L of diluted T4 DNA ligase; 2. Mu.L of 10×T4DNA ligase reaction buffer, the balance being water;
3) The prepared living reaction system is evenly mixed by oscillation and is put into a common PCR instrument after short centrifugation, and the reaction conditions of the PCR instrument are as follows:
；
4) Performing capillary electrophoresis on the reacted product, calculating the area of a main peak, calculating the mass concentration of a substance corresponding to the main peak according to the area and the size of a fragment corresponding to the main peak, and calculating the mass concentration of the substance according to the area of the main peak by using capillary electrophoresis instrument software;
the first group of shorter double-stranded DNA and the second group of longer double-stranded DNA are connected through the catalysis of T4 DNA ligase to form a third group of product double-stranded DNA, and the three main peaks are respectively a first group, a second group and a third group from the short fragment size to the long fragment size; the ligation efficiency was calculated from the quantitative concentration of the substance to determine the T4 DNA ligase activity, wherein the ligation efficiency was calculated as follows:
connection efficiency = mass concentration of the third group substance/(mass concentration of the third group substance + mass concentration of the less concentrated group substance of the first group and the second group) ×100%.
The principle of the invention is as follows:
the invention first prepares the enzymatic reaction substrate of T4 DNA ligase, and forms two double chains containing cohesive ends by annealing two complementary single chains, wherein the cohesive ends of the two double chains are complementary. The two strands are of different lengths to distinguish between subsequent analytical assays. The two double-stranded DNA containing complementary cohesive ends are mutually connected into a longer double-stranded DNA under the enzymatic reaction of T4 DNA ligase, and the T4 DNA ligase in the reaction system is inactivated at high temperature after the reaction is finished. And (3) carrying out capillary electrophoresis on the reaction system, and simultaneously measuring the molar concentration of the three DNA fragments with different lengths by using the analysis software of the capillary electrophoresis result, so as to determine the ratio of the amounts of substances of the fragments. Since two kinds of substrates are required for producing one molecule of long fragment product each, in calculating the ligation efficiency, ligation efficiency=substrate consumption/substrate initial amount×100% =2×product substance mass concentration/2×product substance mass concentration+ (first group substrate substance mass concentration+second group substrate substance mass concentration) ×100%. Since there is a systematic error in the actual operation, the mass concentrations of the substances of the first group of substrates and the second group of substrates do not have a complete concentration agreement, the initial concentration of the two groups of substrates which is the same in the calculation process is the concentration of the substrate having a smaller concentration, that is, the connection efficiency=2×the mass concentration of the product substance/2× (the mass concentration of the product substance+the mass concentration of the substance having a smaller concentration in the first group and the second group of substrates) ×100% =the mass concentration of the substance having a third group/(the mass concentration of the substance having a smaller concentration in the third group+the mass concentration of the substance having a smaller concentration in the first group and the second group) ×100%.
The invention has the following technical effects:
1) The T4 DNA ligase detection kit and the live detection method provided by the invention solve the defects of complex operation and complex steps of the existing live detection method; the phage single-stranded DNA is not required to be used as a reaction substrate, so that the risk of phage residues is avoided, a fluorescent group or a dye is not required to be used, and the need of specially preparing a fluorescence microplate reader or a fluorescence spectrophotometer is avoided.
2) The T4 DNA ligase detection kit and the activity detection method provided by the invention reduce the instrument cost and the consumable cost of the T4 DNA ligase activity detection, thereby reducing the activity detection threshold.
3) The T4 DNA ligase detection kit and the activity detection method provided by the invention shorten the activity detection time, reduce the detection error caused by the low-temperature reaction of the T4 DNA ligase in the activity detection process, and improve the activity detection accuracy.
Drawings
FIG. 1 is a schematic diagram of the length and structure of the substrates and products involved in the enzymatic activity assay reaction.
FIG. 2 is a graph showing the result of capillary electrophoresis performed on one of the enzyme activity assay experiments using a Qsep100 Biopt fully automatic nucleic acid protein analysis system/bioanalyzer.
FIG. 3 is a standard graph plotting ligation efficiencies for four different enzyme activities of T4 DNA ligase.
Detailed Description
The present invention will be further described in detail below with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent, and it is apparent that the described examples are only some of the examples of the present invention, but not all of the examples.
The four single-stranded DNAs used in the invention are synthesized and provided by biological engineering (Shanghai) Inc., and the T4 DNA ligase, the T4 DNA ligase diluent and the T4 DNA ligase reaction buffer used in the invention are provided by Jiangsu kang as century biotechnology Co.
Example 1
The enzyme activity detection kit for T4 DNA ligase of the embodiment comprises the following components which are independently packaged: 1) A reaction substrate material for detecting T4 DNA ligase activity; 2) T4 DNA ligase diluent; 3) T4 DNA ligase reaction buffer.
1) The reaction substrate raw material for detecting the activity of the T4 DNA ligase comprises four single-stranded DNA, wherein the four single-stranded DNA are divided into two groups;
first strip in first group: 5 'gacctctttatgatggaaatctagt'; the nucleotide sequence numbers are SEQ ID NO:1, a step of;
second strip in the first group: 5 'gtcaactagatttccatcataaagaggtc', nucleotide sequence number SEQ ID NO:2, wherein the second 5' end modifies a phosphate group;
first strip in second group:
5'agatacaagggtccagcaatttctcgtgcgggtgctcaactcaac 3'; the nucleotide sequence numbers are SEQ ID NO:3, a step of;
second strips in the second group:
5'tgacgttgagttgagcacccgcacgagaaattgctggacccttgtatct 3', nucleotide sequence number SEQ ID NO:4, wherein the second 5' end modifies a phosphate group.
The four reaction substrate raw materials are all independently packaged in a dark place, and are all 100 mu M.
2) The T4 DNA ligase diluent comprises the following specific components in percentage by weight:
t4 DNA ligase diluent is prepared by the following steps: ddH is used at normal temperature 2 And (3) preparing O, wherein a magnetic stirrer is used for fully and uniformly mixing, standing for 12 hours at 4 ℃ after uniformly mixing, and storing at-25 ℃ to 8 ℃.
3) The 10 xT 4 DNA ligase reaction buffer solution comprises the following specific components in percentage by weight:
10×T4DNA ligase reaction buffer, the preparation method is as follows:
ddH at 4 ℃ 2 And (3) preparing O, wherein a magnetic stirrer is used for fully and uniformly mixing, standing for 1h at 4 ℃ after uniformly mixing, and storing at-25 ℃ to-18 ℃.
Example 2
The enzyme activity of the T4 DNA ligase was determined using the kit of example 1 as follows:
1) Mixing the two single-stranded DNA fragments of the first group according to the molar ratio of 1:1 at the concentration of 100 mu M, mixing the two single-stranded DNA fragments of the second group according to the molar ratio of 1:1 at the concentration of 100 mu M, and respectively carrying out annealing combination in a common PCR instrument; the annealing and combining process is a slow annealing process, the temperature is slowly reduced from 95 ℃ to 45 ℃ and is reduced by 1 ℃ every 10 seconds, so that the two pairs of single chains are completely annealed and combined into two double chains, and the annealing program conditions are set in a common PCR instrument as follows:
；
after annealing and combining, two double-stranded DNA containing cohesive ends are formed respectively, and the product after annealing and combining is diluted by 50 times to 1 mu M, so that a reaction substrate can be obtained.
2) The T4 DNA ligase to be detected is diluted 1000 times by using a T4 DNA ligase diluent, and a detection reaction system is configured as follows:
3) The prepared living reaction system is evenly mixed by oscillation and is put into a common PCR instrument after short centrifugation, and the reaction conditions of the PCR instrument are as follows:
4) And (3) carrying out capillary electrophoresis on the reacted product, calculating the area of a main peak, calculating the mass concentration of the substance corresponding to the main peak according to the area and the size of the fragment corresponding to the main peak, and calculating the mass concentration of the substance according to the area of the main peak and the software of a capillary electrophoresis instrument. As shown in FIG. 1, the first group of shorter double-stranded DNA and the second group of longer double-stranded DNA are connected through the catalysis of T4 DNA ligase to form a third group of product double-stranded DNA, and three main peaks are respectively the first group, the second group and the third group from the short fragment size to the long fragment size. The ligation efficiency was calculated from the quantitative concentration of the substance to determine the T4 DNA ligase activity, wherein the ligation efficiency was calculated as follows:
connection efficiency = mass concentration of third group substance/(mass concentration of third group substance + mass concentration of less concentrated group substance of first group and second group) ×100%
Because of unavoidable errors in the single-stranded DNA synthesis and annealing processes, the concentration of the first and second sets of double-stranded DNA species are not necessarily identical, and in order to make the calculation of the ligation efficiency more accurate, the concentration of the species in the first and second sets of smaller concentration is calculated as the substrate concentration during the calculation.
Taking one detection as an example, after the T4 DNA ligase with the concentration of 10U/mu L is used for dilution, the ligation reaction is carried out, and then the Qsep100 Bioptic full-automatic nucleic acid protein analysis system/biological analyzer is used for capillary electrophoresis, and an electrophoresis chart is shown in FIG. 2, wherein 5 main peaks, 20bp and 1000bp peaks are Alignment markers for calculating the size of fragments in the capillary electrophoresis, 30bp peaks are the first group of double-stranded DNA,52bp peaks are the second group of double-stranded DNA, and 80bp peaks are the third group of double-stranded DNA. Because of instrument errors, the size of the identified fragments deviates slightly from the actual size without affecting the final result. The mass concentration of the fragments shown in each peak measured by the Qsep100 Bioptic fully automatic nucleic acid protein analysis system/bioanalyzer is shown in the following table:
wherein, the connection efficiency is calculated by a connection efficiency calculation formula, and the connection efficiency is= (7.06 nmol/L)/(7.06 nmol/L+7.97 nmol/L) ×100% = 46.97%, and the connection efficiency of the T4 DNA ligase to be detected is 46.97%.
Using this method, standard curves were drawn, enzyme activity assays were performed on 15, 12, 9, 6U/. Mu.L of T4 DNA ligase, the quantitative concentrations of each group of materials were determined and the ligation efficiencies calculated as shown in the following table:
a standard curve is prepared from each enzyme activity T4 DNA ligase and the corresponding ligation efficiency, as shown in FIG. 3, R 2 0.9999.
The foregoing is merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention or direct or indirect application in other related technical fields are included in the scope of the present invention.
Claims (10)
1. An enzyme activity detection kit for T4 DNA ligase is characterized in that the kit comprises a reaction substrate raw material for detecting the activity of the T4 DNA ligase; the reaction substrate raw material comprises four independent single-stranded DNA (deoxyribonucleic acid) which are divided into two groups;
first strip in first group: 5 'gacctctttatgatggaaatctagt'; the nucleotide sequence numbers are SEQ ID NO:1, a step of;
second strip in the first group: 5 'gtcaactagatttccatcataaagaggtc', nucleotide sequence number SEQ ID NO:2, wherein the second 5' end modifies a phosphate group;
first strip in second group:
5'agatacaagggtccagcaatttctcgtgcgggtgctcaactcaac 3'; the nucleotide sequence numbers are SEQ ID NO:3, a step of;
second strips in the second group:
5'tgacgttgagttgagcacccgcacgagaaattgctggacccttgtatct 3', nucleotide sequence number SEQ ID NO:4, wherein the second 5' end modifies a phosphate group.
2. The kit for the enzymatic activity detection of T4 DNA ligase according to claim 1 wherein the kit further comprises a T4 DNA ligase diluent; the T4 DNA ligase diluent comprises the following components: pH buffers, chelating agents, detergents, osmolytes, anti-freeze agents and reducing agents.
3. The enzyme activity detection kit for T4 DNA ligase according to claim 2 wherein the pH of the T4 DNA ligase diluent is 7.5-8.0 and the pH buffer is selected from any one of Tris-HCl, PBS, TAPS; the chelating agent is selected from any one of EDTA-2Na, sodium phosphate and DTPA; the detergent is selected from Tween-20, brij 58, triton X-100 or a combination thereof; the osmotic pressure regulator is selected from KCl and CaCl 2 、NaCl、MgCl 2 Or a combination thereof; the antifreezing agent is selected from glycerol, ethylene glycol or a combination thereof; the reducing agent is selected from DTT, beta-mercaptoethanol, or a combination thereof.
4. The enzyme activity detection kit for T4 DNA ligase according to claim 3, wherein the pH buffer is Tris-HCl, and the content of Tris-HCl is 10 mM-60 mM based on the total amount of the T4 DNA ligase diluent; the chelating agent is EDTA-2Na, and the content of the EDTA-2Na is 0.01 mM-10 mM according to the total amount of the T4 DNA ligase diluent; the detergent is Tween-20, and the content of the Tween-20 is 0.1% -2% (v/v) based on the total amount of the T4 DNA ligase diluent; the osmotic pressure regulator is KCl, and the content of the KCl is 30 mM-80 mM according to the total amount of the T4 DNA ligase diluent; the antifreezing agent is glycerol, and the content of the glycerol is 30% -70% (v/v) based on the total amount of the T4 DNA ligase diluent; the reducing agent is DTT, and the content of the DTT is 0.1 mM-2 mM based on the total amount of the T4 DNA ligase diluent.
5. The enzyme activity detection kit for T4 DNA ligase according to any one of claims 2-4 wherein the kit further comprises a T4 DNA ligase reaction buffer; the reaction buffer is 10×T4DNA ligase reaction buffer, and comprises the following components: pH buffers, concentrates, enzyme activators, reducing agents and ATP.
6. The enzyme activity detection kit for T4 DNA ligase according to claim 5 wherein the pH of the 10 x T4 DNA ligase reaction buffer is 7.5-8.0 and the pH buffer is selected from Tris-HCl, PBS or TAPS; the concentrate is selected from PEG 6000, mannitol or a combination thereof; the enzyme activator is selected from MgCl 2 、MgSO 4 Or a combination thereof; the reducing agent is selected from DTT, beta-mercaptoethanol, or a combination thereof.
7. The enzyme activity detection kit for T4 DNA ligase according to claim 6 wherein the pH buffer is Tris-HCl, the content of Tris-HCl being 300 mM-700 mM based on the total amount of 10 XT 4 DNA ligase reaction buffer; the concentration agent is PEG 6000, and the content of the PEG 6000 is 4% -10% (v/v) based on the total amount of the 10 xT 4 DNA ligase reaction buffer solution; the enzyme activator is MgCl 2 MgCl based on the total amount of 10×T4 DNA ligase reaction buffer 2 The content of (2) is 30 mM-120 mM; the reducing agent is DTT, and the content of the DTT is 1 mM-20 mM according to the total amount of 10 xT 4 DNA ligase reaction buffer solution; the ATP is contained in an amount of 8 mM-20 mM based on the total amount of the 10 xT 4 DNA ligase reaction buffer.
8. The method for measuring the enzyme activity of the T4 DNA ligase by using the kit as claimed in claim 7, wherein the measuring method comprises the following steps:
1) Mixing the two single-stranded DNA fragments of the first group according to the molar ratio of 1:1 at the concentration of 100 mu M, mixing the two single-stranded DNA fragments of the second group according to the molar ratio of 1:1 at the concentration of 100 mu M, and respectively carrying out annealing combination in a common PCR instrument;
2) Diluting the T4 DNA ligase to be detected by 1000 times by using a T4 DNA ligase diluent, wherein a detection reaction system is configured as follows, and each 20 mu L of the detection reaction system contains 5 mu L of a first group of annealing binding products; 5 μl of the second set of annealed binding products; 0.4. Mu.L of diluted T4 DNA ligase; 2. Mu.L of 10×T4 DNA ligase reaction buffer, the balance being water;
3) Mixing the prepared living detection reaction system in an oscillating way, centrifuging for a short time, and then placing the mixture into a PCR instrument for reaction;
4) Performing capillary electrophoresis on the reacted product, calculating the area of a main peak, calculating the mass concentration of a substance corresponding to the main peak according to the area and the size of a fragment corresponding to the main peak, and calculating the mass concentration of the substance according to the area of the main peak by using capillary electrophoresis instrument software;
the first group of shorter double-stranded DNA and the second group of longer double-stranded DNA are connected through the catalysis of T4 DNA ligase to form a third group of product double-stranded DNA, and the three main peaks are respectively a first group, a second group and a third group from the short fragment size to the long fragment size; the ligation efficiency was calculated from the quantitative concentration of the substance to determine the T4 DNA ligase activity, wherein the ligation efficiency was calculated as follows:
connection efficiency = mass concentration of the third group substance/(mass concentration of the third group substance + mass concentration of the less concentrated group substance of the first group and the second group) ×100%.
9. The method according to claim 8, wherein in the step 1), the annealing process is a slow annealing process, the annealing process is slowly cooled from 95 ℃ to 45 ℃ and every 10s is reduced by 1 ℃, so as to ensure that two pairs of single strands are completely annealed and combined into two double strands, and the annealing program conditions are set in a common PCR instrument as shown in the following table:
；
after annealing, two double-stranded DNA containing cohesive ends are formed, and the annealed product is diluted 50-fold to 1. Mu.M.
10. The method according to claim 8, wherein in the step 3), the reaction conditions of the PCR instrument are as follows:
。
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| CN104278090A (en) * | 2014-09-28 | 2015-01-14 | 南京诺唯赞生物科技有限公司 | Method for determining activity of DNA ligase |
| CN108220386A (en) * | 2017-09-24 | 2018-06-29 | 天津强微特生物科技有限公司 | A kind of method of fluoremetry DNA ligase activity |
| CN110408680A (en) * | 2019-06-28 | 2019-11-05 | 山东师范大学 | For detection of alkaline phosphatase, based on ligase amplified reaction catalysis assembling single quantum dot nano-sensor and application |
| CN113061645A (en) * | 2021-04-30 | 2021-07-02 | 上海碧云天生物技术有限公司 | Enzyme activity determination method of DNA ligase |
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| CN104278090A (en) * | 2014-09-28 | 2015-01-14 | 南京诺唯赞生物科技有限公司 | Method for determining activity of DNA ligase |
| CN108220386A (en) * | 2017-09-24 | 2018-06-29 | 天津强微特生物科技有限公司 | A kind of method of fluoremetry DNA ligase activity |
| CN110408680A (en) * | 2019-06-28 | 2019-11-05 | 山东师范大学 | For detection of alkaline phosphatase, based on ligase amplified reaction catalysis assembling single quantum dot nano-sensor and application |
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