CN112746094B - Method for measuring kinetic parameters of pre-steady-state enzymatic reaction of DNA polymerase - Google Patents
Method for measuring kinetic parameters of pre-steady-state enzymatic reaction of DNA polymerase Download PDFInfo
- Publication number
- CN112746094B CN112746094B CN201911038986.0A CN201911038986A CN112746094B CN 112746094 B CN112746094 B CN 112746094B CN 201911038986 A CN201911038986 A CN 201911038986A CN 112746094 B CN112746094 B CN 112746094B
- Authority
- CN
- China
- Prior art keywords
- dna polymerase
- nucleotide
- template
- label
- alexa
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
-
- 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
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
Abstract
The invention relates to a method for measuring kinetic parameters of a pre-steady-state enzymatic reaction of a DNA polymerase, which comprises the following steps: providing a template to be repaired and a substrate, wherein the template to be repaired comprises a template strand and a primer strand which is matched and combined with part of the template strand; respectively mixing a substrate comprising second marked nucleotides with different concentrations with a template to be supplemented and enough DNA polymerase to perform extension reaction, and detecting the fluorescence intensity change of each extension reaction by adopting a stop-flow spectrometry to obtain a plurality of fluorescence intensity change speeds; and obtaining kinetic parameters of the DNA polymerase according to the change speed of the fluorescence intensity and the concentration of the corresponding second marked nucleotide. The accuracy of the dynamic parameters of the pre-steady enzymatic reaction for measuring the DNA polymerase by the method is higher.
Description
Technical Field
The invention relates to a DNA polymerase, in particular to a method for measuring a kinetic parameter of a pre-steady enzymatic reaction of the DNA polymerase.
Background
DNA polymerase (DNA polymerase) is an important enzyme in the replication of DNA by cells and is widely available in almost all organisms. The DNA polymerase can complete the replication of a nucleic acid strand by using DNA or RNA as a template and dNTPs as substrates. Naturally, DNA polymerase and other protein molecules work together to participate in the processes of cell division and genome replication, playing an important role in maintaining the integrity of genetic material.
The diversity of the types, sources, structures and functions of DNA polymerase has led to the discovery of the first DNA polymerase in 1959, and thus, the study of DNA polymerase has never been discontinued. Studies have shown that the DNA polymerase family has numerous members, and that DNA polymerase can be divided into seven subfamilies, A, B, C, D, X, Y and Reverse Transcriptase (RT), based on protein sequence conservation and secondary structural characterization. In recent years, DNA polymerases are widely used in various aspects of biotechnology and scientific research fields, including cDNA synthesis, DNA end modification, DNA sequencing, and the like.
The conventional methods for detecting the activity of DNA polymerase include radioisotope method, PCR method and fluorescent dye incorporation method. Radioisotope method is to incorporate 32P-labeled dNTP catalyzed by DNA polymerase into calf thymus DNA, to filter through TCA precipitation and whatman filter paper, and to measure the radioactive content in acid insoluble substances, thereby calculating the polymerase activity. The radioisotope method is easy to produce radioactive pollution and complex in operation, and is difficult to realize high-flux detection.
The PCR method is to use primer with cy5 fluorescent mark at 5' end to carry out PCR, and to add equal volume of stop solution to stop reaction in different time, wherein the stop solution contains competitive oligonucleotide to prevent the recombination of fluorescent product and template. The results were then verified by electrophoresis using polyacrylamide containing 8M urea.
The fluorescent dye doping method is characterized in that fluorescent dye is doped in a PCR reaction system, the fluorescence intensity of the reaction system 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, a standard curve can be drawn by using polymerase with known enzyme activity according to the characteristic, and the initial rate of the unknown polymerase reaction is brought into the standard curve, so that the enzyme activity of the unknown polymerase can be obtained.
Although the PCR method and the fluorescent dye doping method can be used for detecting the activity of the DNA polymerase to avoid radioactive pollution, the PCR method and the fluorescent dye doping method are both kinetic parameters of the detected DNA polymerase under the steady-state condition, and the enzyme activity of the DNA polymerase under the steady-state condition is evaluated. The kinetic parameters for the catalytic reaction of DNA polymerase under pre-steady state conditions have not been detected nor assessed as the enzymatic activity of DNA polymerase under pre-steady state conditions.
Disclosure of Invention
Based on this, it is necessary to provide a method for measuring the kinetic parameters of the pre-steady-state enzymatic reaction of DNA polymerase.
A method for determining kinetic parameters of a pre-steady-state enzymatic reaction of a DNA polymerase comprising the steps of:
providing a template to be filled in, wherein the template to be filled in comprises a template strand and a primer strand which is matched and combined with part of the template strand, the template to be filled in is provided with a first mark, the substrate comprises a nucleotide with a second mark, the second mark and the first mark can generate fluorescence resonance energy transfer, and only one nucleotide which is not matched and combined with the primer strand in the template strand can be complementarily matched with the nucleotide with the second mark;
mixing substrates comprising the second marked nucleotide with different concentrations with the template to be supplemented and enough DNA polymerase respectively to perform extension reactions of the primer chains of the second marked nucleotide with different concentrations, and detecting fluorescence intensity changes of each extension reaction by adopting a stop-flow spectrometry to obtain a plurality of fluorescence intensity change speeds; a kind of electronic device with high-pressure air-conditioning system
And obtaining the pre-steady enzymatic reaction kinetic parameters of the DNA polymerase according to the change speeds of the fluorescence intensities and the concentrations of the corresponding second marked nucleotides.
According to the method for measuring the kinetic parameters of the pre-steady-state enzymatic reaction of the DNA polymerase, the first mark and the second mark in the extension reaction of the template to be supplemented are adopted to generate fluorescence resonance energy transfer, and the rapid residence technology is combined to detect the change of the fluorescence intensity in the extension reaction, so that the kinetic parameters of the pre-steady-state enzymatic reaction of the DNA polymerase are obtained. The rapid reaction residence technology can ensure that samples are rapidly mixed uniformly, can detect the change of fluorescence intensity in millisecond-second time except dead time, and can also ensure that the fluorescence detection is more accurate, so that the kinetic parameters of the pre-steady state of the DNA polymerase can be detected, and the result accuracy is higher.
In one embodiment, the pre-steady state enzymatic reaction kinetic parameters of the DNA polymerase include a dissociation constant and a binding constant of the nucleotide having the second label.
In one embodiment, the step of obtaining the kinetic parameters of the DNA polymerase according to the plurality of rates of change of the fluorescence intensity and the corresponding concentrations of the second labeled nucleotides comprises:
performing linear fitting on a plurality of the change speeds of the fluorescence intensity and the corresponding nucleotide concentrations with the second marks to obtain a linear formula, and combining the linear formula with k obs ＝k on [L]+k off Aligning to obtain a dissociation constant and a binding constant of the nucleotide having the second label, wherein the k obs ＝k on [L]+k off In (k) obs For the rate of change of fluorescence intensity, [ L ]]To have the concentration of the second labeled nucleotide, k on K being the binding constant of the nucleotide having the second label off Is the dissociation constant of the nucleotide having the second label.
In one embodiment, the first label is one of a fluorescence donor group and a fluorescence acceptor group and the second label is the other of a fluorescence donor group and a fluorescence acceptor group.
In one embodiment, the fluorescent donor group is selected from one of Alexa flexo 350, alexa flexo 448, alexa flexo 546, alexa flexo 555, alexa flexo 568, alexa flexo 594, and Alexa flexo 647;
and/or the fluorescent acceptor group is selected from one of Cy5, alexa Fluro 488, alexa Fluro 546, alexa Fluro 555, alexa Fluro 568, alexa Fluro 594 and Alexa Fluro 647.
In one embodiment, the first labeled nucleotide is attached to the template to be leveled, and the distance between the first labeled nucleotide and the nucleotide on the template to be leveled and the nucleotide with the second label is between 0nm and 560nm.
In one embodiment, the primer strand has a first label; and/or, when the DNA polymerase has 3' -5' exonuclease activity, the base at the 3' end of the primer strand is thio-modified.
In one embodiment, the substrate further comprises unlabeled nucleotides having a base different from the base of the second labeled nucleotide.
In one embodiment, the base sequence of the template strand is shown in SEQ ID No. 1; and/or
The base sequence of the primer chain is shown as SEQ ID No. 2.
In one embodiment, the nucleotide having the second tag has the formula:
drawings
FIG. 1 is an excitation spectrum and an emission spectrum of Alexa Fluro 488;
FIG. 2 is an excitation spectrum and an emission spectrum of Alexa Fluro-647;
FIG. 3 is an excitation spectrum and an emission spectrum of Cy 5;
FIG. 4 is a PAGE electrophoresis of example 1.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. Some embodiments of the invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
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 herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The method for measuring the kinetic parameters of the pre-steady-state enzymatic reaction of the DNA polymerase according to one embodiment comprises the steps S110 to S140.
Step S110, providing a template and a substrate to be leveled.
Specifically, the template to be repaired comprises a template strand and a primer strand which is matched and combined with part of the template strand, and the template to be repaired is provided with a first mark. The substrate comprises a nucleotide having a second label, the second label and the first label being capable of fluorescence resonance energy transfer. Only one of the nucleotides of the template strand that is not paired with the primer strand is complementarily paired with the nucleotide having the second label.
In general, the template strand is 15bp to 50bp in length, and the primer strand is 50bp to 45bp in length.
The primer strand has a first label, which is a fluorescent group. The first label is located at the 3 'end of the primer strand and is linked to the base at the 3' end of the primer strand. It will be appreciated that the location of the first label is not limited to the 3' end of the primer strand, but may be other locations of the template to be filled in. For example, the first label may be located at the 5 'end of the template strand and linked to the base at the 5' end of the template strand.
Further, the base at the 3' -end of the primer strand is thio-modified. After being thio-modified, O in the phosphodiester bond between original bases is replaced with S. The thio modification is to prevent the cleavage of the nucleotide to which the first label is attached at the 3' -end of the primer strand by a DNA polymerase having 3' -5' exonuclease activity. Of course, in other embodiments, other modifications may be used to modify the 3 'end of the primer strand to avoid cleavage of the 3' end of the primer strand by a DNA polymerase having 3'-5' exonuclease activity.
In this embodiment, the base sequence of the template strand is shown in SEQ ID No.1, and the base sequence of the primer strand is shown in SEQ ID No. 2. A first label is attached to thymine at the first of the primer strand in the 3 'to 5' direction. Specifically, the base sequence shown in SEQ ID No.1 is: 5'-TTTTTTGCAAGGCTGGTCGGTCAGTC-3'. The base sequence shown in SEQ ID No.2 is: 5'-GACTGACCGACCAGCCT T G-3'. Here, "T" means that the 3' -end has improved cleavage resistance after O in the phosphodiester bond between T and between T and G is replaced with S and O in the phosphodiester bond between T, T and G is replaced with S. Of course, in other embodiments, O in the phosphodiester linkage between the first G and the first T, which may also be the 3' end, is replaced with S; or O in the phosphodiester linkage between the first T and the second T is replaced with S.
Of course, in other embodiments, the base sequence of the template strand is not limited to that shown in SEQ ID No.1, and may be any other base sequence that corresponds to the template. Accordingly, the base sequence of the primer strand is not limited to that shown in SEQ ID No.2, and the base sequence of the primer strand may be adjusted according to the design of the template strand.
Further, the first label is one of a fluorescence donor group and a fluorescence acceptor group, and the second label is the other of a fluorescence donor group and a fluorescence acceptor group. Specifically, the fluorescent donor group is selected from one of Alexa flexo 488, alexa flexo 546, alexa flexo 555, alexa flexo 568, alexa flexo 594 and Alexa flexo 647. The fluorescent acceptor group is selected from one of Cy5, alexa Fluro 488, alexa Fluro 546, alexa Fluro 555, alexa Fluro 568, alexa Fluro 594 and Alexa Fluro 647.
In one embodiment, the template to be filled with the first labeled nucleotide is attached at a distance of 0nm to 560nm from the complementary paired nucleotide on the template to be filled with the second labeled nucleotide.
Specifically, the nucleotide having the second label includes a second label, a base (purine base or pyrimidine base), deoxyribose, and phosphate; the second label, phosphate, deoxyribose and base are linked by chemical bonds in sequence. Wherein the phosphoric acid is not limited to the triphosphate having three phosphate groups, but may be a phosphoric acid having other number of phosphate groups, such as monophosphate, hexaphosphate, etc. The nucleotide with the second label is integrated onto the template to be filled under the action of the DNA polymerase, and of course, the specific position of the second label to be integrated onto the template to be filled is required to enable fluorescence resonance energy transfer of the first label and the second label.
In one embodiment, the nucleotide having the second label is deoxycytosine ribose hexaphosphate having the second label. Further, the nucleotide having the second label further comprises a linker arm through which the second label is linked to the phosphate. The linker arm is typically an alkyl group for increasing the distance between the second label and the phosphate and reducing the binding of the second label to the template strand of the nucleotide having the second label.
In this embodiment, the structure of the nucleotide having the second label is as follows:
in one embodiment, the substrate further comprises unlabeled nucleotides. Specifically, unlabeled nucleotides may be provided according to the base sequence of the template strand. Further, the base of the unlabeled nucleotide is different from the base of the nucleotide having the second label. For example, if the base having the second labeled nucleotide is cytosine, the base of the unlabeled nucleotide is another base other than cytosine.
In one embodiment, the first label is Alexa fluor 488 and the second label is Cy5. The fluorescent dye Cy5 has similar excitation and emission spectral ranges to Alexa Fluor 647, but is tens of times different in price. The excitation spectra and emission spectra of Alexa Fluro-488, alexa Fluro-647 and Cy5 are shown in FIGS. 1 to 3 in this order.
And step S120, mixing the template to be filled, the substrate and the DNA polymerase to perform the extension reaction of the primer chain, and obtaining the change speed of the fluorescence intensity of the extension reaction by adopting a stopped flow spectrometry.
In this embodiment, the conditions for the extension reaction are: 25-37 ℃ for 5-20 min. Preferably, the conditions of the extension reaction are: x is 30-37 ℃ for 5-10 min. Of course, in other embodiments, the extension conditions may be designed based on the particular template strand, primer strand, and substrate.
The system of the extension reaction comprises: 0.5-1. Mu.M of template to be filled in, 100-1000. Mu.M of nucleotide with second label and 0.1-0.5. Mu.M of DNA polymerase.
In this embodiment, the DNA polymerase is Phi29 DNA polymerase. Of course, the DNA polymerase is not limited to Phi29 DNA polymerase, but may be other DNA polymerases in the art.
Specifically, adding a template to be supplemented, a substrate comprising a nucleotide with a second label and a sufficient amount of DNA polymerase into a stopped flow spectrometer for extension reaction; detecting the fluorescence intensity change of the first mark or the second mark by the flow stopping spectrometer, collecting the fluorescence intensity change data of the first mark or the second mark, performing nonlinear regression on the collected fluorescence intensity data of the first mark or the second mark by the flow stopping spectrometer through an exponential equation, and fitting a dynamics curve; and then outputting the change speed of the fluorescence intensity of the extension reaction according to the kinetic curve.
When the amount of the substrate is insufficient, the same DNA polymerase catalyzes the extension reaction at a different rate, and the resulting fluorescence intensity varies at a different rate. Further, under the conditions of the same concentration of the template to be filled and the same concentration of the DNA polymerase, a plurality of fluorescence intensity change speeds are obtained by detecting the fluorescence intensity change of a plurality of different concentrations (without excessive) of the nucleotide with the second label by using a stopped flow spectrometer.
Step S130, obtaining kinetic parameters of the DNA polymerase according to a plurality of fluorescence intensity change speeds.
In a DNA polymerase-promoted DNA amplification reaction, the DNA polymerase is first bound to a primer-template complex (i.e., the template to be filled in as mentioned above), and then free deoxyribonucleotides are integrated. In most DNA polymerases, the deoxyribonucleotide to be bound enters the pre-insertion site (pre-insertion site) subtended by the template strand and then enters the insertion site (insertion site); the DNA polymerase then catalyzes deoxyribonucleotide integration, primer strand extension by one base and pyrophosphate binding of one molecule near the active site. With the newly formed primer strand, the DNA polymerase end moves out of the deoxyribonucleotide insertion site, the alpha-phosphate bond breaks, and pyrophosphate is released. The rate at which the DNA polymerase binds to and dissociates dntps (or dNTP analogues) and the rate at which the product is released thus together determine the rate of polymerization of the DNA polymerase.
In this embodiment, the parameters of the pre-steady state enzymatic reaction kinetics of the DNA polymerase include the dissociation constant and the binding constant of the nucleotide having the second label.
Specifically, a plurality of fluorescence intensity change speeds and the corresponding nucleotide concentrations with the second marks are subjected to linear fitting to obtain a linear formula; then the curve formula is combined with k obs ＝k on [L]+k off Alignment is performed to obtain the dissociation constant and the binding constant of the nucleotide having the second label. Wherein k is obs ＝k on [L]+k off In [ L ]]Refers to the concentration, k, of the nucleotide having the second label obs The change speed, k of the fluorescence intensity obtained by the stop-flow spectrometer on Refers to the binding constant, k, of the nucleotide with the second label off Refers to the dissociation constant of the nucleotide with the second label. For the same DNA polymerase and a specific nucleotide substrate, k is the same on And k off Is unchanged.
According to the method for measuring the kinetic parameters of the pre-steady-state enzymatic reaction of the DNA polymerase, the first mark and the second mark generate fluorescence resonance energy transfer in the extension reaction of the template to be supplemented, and the rapid retention technology is combined to detect the change of the fluorescence intensity in the extension reaction when the substrate quantity is insufficient, so that the change speed of the fluorescence intensity under different substrate concentration conditions is obtained, and the kinetic parameters of the pre-steady-state enzymatic reaction of the DNA polymerase are obtained. The rapid reaction residence technology can be used for rapidly and uniformly mixing samples, detecting the fluorescence intensity change in the millisecond-second time except dead time, can be used for measuring the forward steady-state enzymatic reaction kinetic parameters of the DNA polymerase, and has more accurate fluorescence detection and higher accuracy of the measured forward steady-state enzymatic reaction kinetic parameters of the DNA polymerase.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following is a detailed description of specific embodiments. The following examples are not specifically described but do not include other components than the unavoidable impurities. The drugs and apparatus used in the examples are all routine choices in the art, unless specifically indicated. The experimental methods without specific conditions noted in the examples were carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer.
Example 1
(1) The extension reaction systems corresponding to the control group, the experimental group 1 and the experimental group 2 shown in table 1 were prepared. In table 1: the template of the template to be leveled is synthesized by Invitrogen corporation; the base sequence of the template strand of the template to be repaired is shown as SEQ ID No.1, the base sequence of the primer strand of the template to be repaired is shown as SEQ ID No.2, alexa Fluro 488 is connected to the first thymine at the 3 'end of the primer strand, and O in the phosphodiester bond between the first thymine and the second thymine at the 3' end of the primer strand is replaced by S (point thio modification). Cy5-dC6P is a nucleotide with a second label, and the structural formula of Cy5-dC6P is:
TABLE 1
(2) And respectively reacting the extension reaction systems corresponding to the control group, the experimental group 1 and the experimental group 2 for 10min at the temperature of 30 ℃ to obtain reaction products of each group.
(3) After adding 6xDNA loading to each group of reaction products, the reaction products were loaded into 15% TBU polyacrylamide gel (PAGE gel) for electrophoresis, wherein the electrophoresis conditions were 180v and 1.5h. After electrophoresis, the PAGE gel is soaked in SYBR Gold dye solution and slowly and horizontally oscillated for about 20min. Detection was then performed using Azure Biosystems imaging. The detection results are shown in FIG. 4. In FIG. 4, lane M is DNA marker, lane 1 is control, lanes 2 and 3 are both experimental 1, and lane 4 is experimental 2.
As can be seen from FIG. 1, cy5-dC6P can be used as a substrate for DNA extension reaction.
Example 2
(1) The system shown in Table 2 was prepared, wherein 7 parts of system 1 and 1 part of each of systems 2 to 8 were prepared.
TABLE 2
(2) 1 part of system 1 and 1 part of system 2 were combined as a first set for reaction and detection: 1 part of system 1 was injected into the syringe 1 of a stopped-flow spectrometer (SX 20 stopped-flow spectrometer of UK light applied Physics (Applied Photophysics)) and 1 part of system 2 was injected into the syringe 2 of the stopped-flow spectrometer, the reaction was carried out after single mixing (mixing of reactants according to 1:1) with the injected stopped-flow spectrometer, and the change in the fluorescence intensity of Alexa Fluro-488 was detected to obtain the change speed of the fluorescence intensity of the first group of Cy5-dC 6P. Wherein the reaction condition is that the temperature of the circulating water bath is 30 ℃ for 5min. The setting of the stay spectrometer is as follows: band, 8nm; pathlength,2mm; the excitation slit is 2nm; the excitation wavelength of Alexa Fluro 488 is selected to be 493nm,Alexa Fluro 488 and the emission wavelength of Alexa Fluro 488 is selected to be bandpass filtered XF3084 (510 nm-570 nm, omega Optical Co., U.S.A.).
1 part of system 1 and 1 part of system 3 were combined as a second group, 1 part of system 1 and 1 part of system 4 were combined as a third group, 1 part of system 1 and 1 part of system 5 were combined as a fourth group, 1 part of system 1 and 1 part of system 6 were combined as a fifth group, 1 part of system 1 and 1 part of system 7 were combined as a sixth group, 1 part of system 1 and 1 part of system 8 were combined as a seventh group, and a stopped-flow spectrometer was injected, and reaction and detection were performed using the same reaction conditions and detection conditions as the first group, to obtain the fluorescence intensity change rate of Cy5-dC6P of each group.
(3) And (3) fitting a curve according to the change speeds of the fluorescence intensity of the plurality of fluorescence Cy5-dC6P obtained in the step (2) and the corresponding concentration of Cy5-dC6P to obtain a linear formula. The formula and formula: k (k) obs ＝k on [L]+k off Performing comparison to obtain k of Phi29 polymerase on And k off 。
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> Shenzhen Qinghua university institute
Anxuyuan Biotechnology (Shenzhen) Co., Ltd.
<120> method for measuring kinetic parameters of a pre-steady-state enzymatic reaction of a DNA polymerase
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ttttttgcaa ggctggtcgg tcagtc 26
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gactgaccga ccagccttg 19
Claims (8)
1. A method for determining kinetic parameters of a pre-steady-state enzymatic reaction of a DNA polymerase, comprising the steps of:
providing a template to be filled in, wherein the template to be filled in comprises a template strand and a primer strand which is matched and combined with part of the template strand, the template to be filled in is provided with a first mark, the substrate comprises a nucleotide with a second mark, and only one nucleotide which is not matched and combined with the primer strand in the template strand can be matched and combined with the nucleotide with the second mark in a complementary way; the nucleotide with the second mark is integrated on the template to be supplemented under the action of DNA polymerase, and the integration of the nucleotide with the second mark on the specific position of the template to be supplemented can enable the first mark and the second mark to generate fluorescence resonance energy transfer;
mixing insufficient amounts of substrates comprising the second labeled nucleotides with different concentrations with the template to be filled and sufficient amounts of DNA polymerase respectively to perform extension reactions of the primer chains of the second labeled nucleotides with different concentrations, and detecting fluorescence intensity changes of each extension reaction by adopting a stop-flow spectrometry to obtain a plurality of fluorescence intensity change speeds; a kind of electronic device with high-pressure air-conditioning system
Obtaining a pre-steady enzymatic reaction kinetic parameter of the DNA polymerase according to the change speeds of the fluorescence intensities and the corresponding concentrations of the nucleotides with the second marks;
the kinetic parameters of the pre-steady state enzymatic reaction of the DNA polymerase are the dissociation constant and the binding constant of the nucleotide having the second label;
the step of obtaining kinetic parameters of the DNA polymerase according to the plurality of rates of change of the fluorescence intensity and the corresponding concentrations of the second labeled nucleotides comprises:
performing linear fitting on a plurality of the change speeds of the fluorescence intensity and the corresponding nucleotide concentrations with the second marks to obtain a linear formula, and combining the linear formula with k obs ＝k on [L]+k off Aligning to obtain a dissociation constant and a binding constant of the nucleotide having the second label, wherein the k obs ＝k on [L]+k off In (k) obs For the rate of change of fluorescence intensity, [ L ]]To have the concentration of the second labeled nucleotide, k on K being the binding constant of the nucleotide having the second label off Is the dissociation constant of the nucleotide having the second label.
2. The method for determining a kinetic parameter of a pre-steady state enzymatic reaction of a DNA polymerase according to claim 1, wherein the first label is one of a fluorescence donor group and a fluorescence acceptor group and the second label is the other of a fluorescence donor group and a fluorescence acceptor group.
3. The method for determining the kinetic parameters of the pre-steady-state enzymatic reaction of a DNA polymerase according to claim 2, wherein the fluorescent donor group is selected from one of Alexa flexo 350, alexa flexo 448, alexa flexo 546, alexa flexo 555, alexa flexo 568, alexa flexo 594 and Alexa flexo 647;
and/or the fluorescent acceptor group is selected from one of Cy5, alexa Fluro 488, alexa Fluro 546, alexa Fluro 555, alexa Fluro 568, alexa Fluro 594 and Alexa Fluro 647.
4. The method for measuring kinetic parameters of a pre-steady-state enzymatic reaction of a DNA polymerase according to claim 1, wherein the first labeled nucleotide is attached to the template to be filled in, and the distance between the first labeled nucleotide and the nucleotide on the template to be filled in and the nucleotide with the second label is 0nm to 560nm.
5. The method for determining a kinetic parameter of a pre-steady state enzymatic reaction of a DNA polymerase according to claim 1, wherein the primer strand has a first label; and/or, when the DNA polymerase has 3' -5' exonuclease activity, the base at the 3' end of the primer strand is thio-modified.
6. The method of measuring a kinetic parameter of a pre-steady state enzymatic reaction of a DNA polymerase according to claim 1, wherein the substrate further comprises unlabeled nucleotides having a base different from the base of the nucleotide having the second label.
7. The method for determining kinetic parameters of a pre-steady-state enzymatic reaction of a DNA polymerase according to claim 1, wherein the base sequence of the template strand is shown in SEQ ID No. 1; and/or
The base sequence of the primer chain is shown as SEQ ID No. 2.
8. The method for determining a kinetic parameter of a pre-steady state enzymatic reaction of a DNA polymerase according to claim 1, wherein the nucleotide having the second label has the structural formula:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911038986.0A CN112746094B (en) | 2019-10-29 | 2019-10-29 | Method for measuring kinetic parameters of pre-steady-state enzymatic reaction of DNA polymerase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911038986.0A CN112746094B (en) | 2019-10-29 | 2019-10-29 | Method for measuring kinetic parameters of pre-steady-state enzymatic reaction of DNA polymerase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112746094A CN112746094A (en) | 2021-05-04 |
| CN112746094B true CN112746094B (en) | 2023-06-13 |
Family
ID=75640131
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911038986.0A Active CN112746094B (en) | 2019-10-29 | 2019-10-29 | Method for measuring kinetic parameters of pre-steady-state enzymatic reaction of DNA polymerase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112746094B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010036359A2 (en) * | 2008-09-25 | 2010-04-01 | Allelogic Biosciences Corporation | Methods of polymerase activity assay |
| AU2011202896A1 (en) * | 2004-06-01 | 2011-07-07 | Abbott Diagnostics Scarborough, Inc. | Recombinase Polymerase Amplification |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2606149B1 (en) * | 2010-08-20 | 2017-08-02 | Life Technologies Corporation | Polymerase assay with a fret substrate |
| WO2013096692A1 (en) * | 2011-12-21 | 2013-06-27 | Illumina, Inc. | Apparatus and methods for kinetic analysis and determination of nucleic acid sequences |
| EP3411494A4 (en) * | 2015-11-18 | 2020-03-11 | Kalim U. Mir | Super-resolution sequencing |
-
2019
- 2019-10-29 CN CN201911038986.0A patent/CN112746094B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2011202896A1 (en) * | 2004-06-01 | 2011-07-07 | Abbott Diagnostics Scarborough, Inc. | Recombinase Polymerase Amplification |
| WO2010036359A2 (en) * | 2008-09-25 | 2010-04-01 | Allelogic Biosciences Corporation | Methods of polymerase activity assay |
Non-Patent Citations (5)
| Title |
|---|
| A single-molecule FRET sensor for monitoring DNA synthesis in real time;Carel Fijen等;Phys Chem Chem Phys;第19卷(第6期);4222-4230 * |
| Measurement of DNA polymerase incorporation kinetics of dye-labeled nucleotides using total internal reflection fluorescence microscopy;Matthew T Walsh等;Biochemistry;第54卷(第26期);4019-4021 * |
| RecQ解旋酶的动力学机理研究;丁秀艳;中国博士学位论文全文数据库基础科学辑(第10期);A006-17 * |
| Single-molecule measurements of synthesis by DNA polymerase with base-pair resolution;Thomas D Christian等;Proc Natl Acad Sci U S A;第106卷(第50期);21109-21114 * |
| 基于氧化石墨烯和阳离子共轭聚合物的端粒酶活性分析;周晓毓;中国优秀硕士学位论文全文数据库工程科技Ⅰ辑(第1期);B014-185 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112746094A (en) | 2021-05-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11891663B2 (en) | Methods, compositions, and kits comprising linker probes for quantifying polynucleotides | |
| Fuller et al. | Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array | |
| US10273473B2 (en) | Method of normalizing biological samples | |
| JP2021118697A (en) | Digital specimen analysis | |
| ES2764096T3 (en) | Next generation sequencing libraries | |
| JP5354216B2 (en) | Nucleic acid probe for nucleic acid measurement, nucleic acid probe set, and target nucleic acid measurement method using them | |
| CN101346472B (en) | Sequencing and genotyping using reversibly 2'-modified nucleotides | |
| US9096895B2 (en) | Method for quantification of small RNA species | |
| CN101965410A (en) | System and method for improved processing of nucleic acids for production of sequencable libraries | |
| CN101528945A (en) | 2'-terminator related pyrophosphorolysis activated polymerization | |
| US20060057595A1 (en) | Compositions, methods, and kits for identifying and quantitating small RNA molecules | |
| EP0665293A2 (en) | Method of determining base sequence of nucleic acid | |
| CN104640984A (en) | Photocoupling method using probe containing photoresponsive nucleic acids | |
| US20060003337A1 (en) | Detection of small RNAS | |
| CA3118607A1 (en) | Method for sequencing polynucleotides | |
| CN112746094B (en) | Method for measuring kinetic parameters of pre-steady-state enzymatic reaction of DNA polymerase | |
| CN105950755A (en) | Method for detecting microRNA based on split-type recognition mode and cascade signal amplification strategy | |
| Einat | Methodologies for high-throughput expression profiling of microRNAs | |
| CN108646014A (en) | The method of fluoroscopic examination platelet derived growth factor based on aptamer conformation variation | |
| WO2023116575A1 (en) | Adapter for characterizing target polynucleotide, method, and use thereof | |
| CN106916882A (en) | Method for dual allele-specific polymerase chain reaction of genotype identification chip for identifying polymorphism of nucleotide gene | |
| WO2023122499A1 (en) | Periodate compositions and methods for chemical cleavage of surface-bound polynucleotides | |
| WO2022129439A1 (en) | Methods, systems and compositions for nucleic acid sequencing | |
| AU2022419500A1 (en) | Periodate compositions and methods for chemical cleavage of surface-bound polynucleotides | |
| JP2006006212A (en) | Reagent for quantitative determination of nucleic acid and method for quantitative determination of nucleic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |