34 results about "T4 polynucleotide kinase" patented technology

The invention provides a kit and method for constructing a DNA library. The kit comprises an enzymic reagent, a buffer solution and a joint combination, wherein the enzymic reagent comprises a first enzyme mixed liquid, T4DNA ligase and a second enzyme mixed liquid, and the first enzyme mixed liquid is formed by mixing T4 Polynucleotide Kinase, T4DNA polymerase, a Klenow fragment and rTaq enzyme; the second enzyme mixed liquid is a 2X KAPA HiFi HotStart pre-mixed solution; the buffer solution comprises a first buffer solution and a second buffer solution, and the first buffer solution is formed by mixing a 10X T4DNA ligase buffer solution, dNTP mixed liquor and ATP; the second enzyme mixed liquid is formed by mixing ATP, Tris-HCL, MgCl2, PEG8000, Tween 20 and dithiothreitol; the joint combination comprises a joint sequence group and a tag sequence group. According to the kit, the library construction cost can be reduced and the amplification efficiency is improved.

The invention discloses a silver nanocluster fluorescent probe based T4 polynucleotide kinase activity detecting method. The silver nanocluster fluorescent probe based T4 polynucleotide kinase activity detecting method is characterized in that a marker-free 'turn-off' type fluorescent probe is manufactured for detecting activity of T4 PNK. A DNA chain for preparing silver nanocluster template is S1(DNA S1), and a DNA chain containing [3'-G4(TG4)2TG3] sequence is S2(DNA S2), and the two chains are hybridized; under action of a G-rich sequence, a silver nanocluster can generate strong red fluorescence, and 5-hydroxyl terminal of the double chains DNA is phosphorylated through T4 PNK, so that a 5-phosphoryl group terminal is generated; the 5-phosphoryl group terminal can be quickly distinguished and degraded by gamma exonuclease, so that two DNA chains are separated, the fluorescence signal is quickly weakened to generate a signal which is in inverse proportion with T4 PNK concentration.Therefore, the silver nanocluster fluorescent probe based T4 polynucleotide kinase activity detecting method can be used for precisely detecting activity of T4 PNK; and the detecting sensitivity of the method can be 0.01 U/mL according to the strength of the DNA-AgNCs fluorescence signal.

The invention discloses a novel method for beacon-free detection of T4 PNKP (T4 polynucleotide kinase)/phosphatase and an inhibitor of T4 PNKP/phosphatase on the basis of fluorescent copper nanoparticles. The method is implemented according to steps as follows: 1), a hairpin probe modified by 3'-terminal phosphorylation is designed, and 5' terminal is a sequence containing a base T; 2), the probe is diluted to the concentration being 100-1,000 nM with a Tris-HCl buffer solution, certain quantities of T4 PNKP, Klenow Fragment and dNTPs are mixed with the probe, and the mixture reacts at the temperature of 37 DEG C for 40-150 minutes; 3), the reaction liquid is diluted to form an MOPS buffer system, and copper sulfate and sodium ascorbate are added and react for 1-30 minutes at the room temperature; 4), the fluorescence strength of the finally obtained reaction liquid is detected with a fluorescence instrument, and a corresponding relation between the fluorescence strength of the system and T4 PNKP concentration is obtained. According to the method, the operation is simple, the cost is low, the sensitivity is high, the selectivity is good, T4 PNKP and the inhibitor can be quantitatively detected and screened, and a potential application value is provided for establishment of other terminal modification enzyme detection methods and molecular biological diagnosis.

The invention discloses a method for quantitatively determining activity of T4 polynucleotide kinase. The method for quantitatively determining the activity of T4 polynucleotide kinase comprises the following steps: in a reaction system used for phosphorylation, adding ATP by taking oligonucleotide as a substrate for phosphorylating an end 5' of oligonucleotide by utilizing T4 polynucleotide kinase, so as to generate phosphorylated oligonucleotide; then adding a luciferase reporter gene system into the reaction system, wherein luciferase in the luciferase reporter gene system utilizes residual ATP in the reaction system to oxidize luciferin to form oxygenated luciferase and produce chemiluminiscence at the same time; and detecting numerical value of luminescence, and deriving activity degree of T4 polynucleotide kinase by virtue of detection results, wherein the activity degree of T4 polynucleotide kinase is in negative correlation with luminous quantity. Compared with the prior art, the method for quantitatively determining the activity of T4 polynucleotide kinase has the advantages that no radioactive contamination is produced, operation is easy, and quantitative detection analysis can be carried out on the activity of T4 polynucleotide kinase.

The invention belongs to the technical field of molecular biology and particularly relates to a method for performing DNA terminal repair/dA addition by adopting a one-step method and application thereof. The method adopts an enzyme system of T4DNA polymerase, Taq DNA polymerase and T4 polynucleotide kinase. The time required by the DNA terminal repair/dA addition in the method is shortened from one and a half hours to 40 minutes, and the reagent consumable consumption is reduced due to the fact that a DNA purification step is omitted, the cost is saved, DNA loss caused by purification is avoided, the preparation efficiency of a DNA library is improved, and the lowest DNA quality required by preparation of the DNA library is reduced.

The invention discloses a quantitative detection method of T4 polynucleotide kinase. The quantitative detection method comprises the steps of: designing a sulfhydrylated single-stranded DNA probe a, aprobe b and a probe c of which a 5'end is modified with amino, and fixing the probe a on the surface of a gold electrode through a gold-sulfhydryl bond, wherein the hydroxyl at the 5'end of the probea can be catalyzed and phosphorylated by T4 polynucleotide kinase, the DNA probe a is then hybridized with the probe b and the probe c, and the 3 'end of the probe c and a phosphate group at the 5' end of the probe a are subjected to a ligation reaction under the action of T4 DNA ligase; performing heating denaturating treatment, wherein the probe c is still retained on the surface of the electrode, the amino group at the tail end of the probe c can further capture the silver nanoparticles, and finally, the quantitative detection result of the T4 polynucleotide kinase is obtained by detectingthe stripping volt-ampere signal of the silver nanoparticles. The method has the characteristics of the high sensitivity, the good selectivity, the rapidness and the low cost, and the detection limitis 0.01 U/mL.

The invention relates to a novel phosphorylated adenosine acylase and a preparation method and application thereof, prokaryotic inducible expression plasmid of Adenylase (PF0353) and T4 PNK (T4 Polynucleotide Kinase) fusion enzyme is constructed, the target fusion enzyme is obtained through expression and purification, the enzyme is high in water solubility and simple in expression and purification, the obtained fusion enzyme has the activity of T4 polynucleotide kinase and adenosine acylase at the same time, the 5'-hydroxy DNA can be subjected to the direct catalytic reaction in the presence of ATP to obtain the 5'-adenylate acylation DNA by using the one-step method, and the method for preparing the adenylate acylation DNA by using the fusion enzyme has advantages of low cost, high reaction efficiency, simpleness, convenience and rapidness, and can be flexibly used by the molecular biological technology.

The invention relates to expression and purification of T4 bacteriophage polynucleotide kinase (T4 PNK) and particularly relates to soluble expression and a purification method of the T4 PNK, belonging to the field of biochemistry. According to the invention, a T4 PNK gene is cloned into a pGEX vector through vector construction, the T4 polynucleotide kinase and GST undergo fused expression under the condition of low temperature, then, the protein expression level reaches 15%, and the solubility reaches 95%.

The invention relates to the field of gene sequencing, in particular to a construction method for a short RNA fragment library, a kit and application. The construction method comprises the following steps of carrying out pre-phosphorylation and dephosphorylation treatment on a short RNA fragment sample, so that a 3' terminal phosphate group in a short RNA fragment is modified into a hydroxyl group, and a 5' terminal hydroxyl group is modified into a phosphate group to obtain a first product; then connecting a 3' linker to obtain a first linker product; then carrying out dephosphorylation treatment to obtain a second product of which a 5' terminal is a phosphate group; then connecting a 5' linker to obtain a second linker product; and then carrying out reverse transcription and amplification to obtain the short RNA fragment library. The kit comprises T4 polynucleotide kinase and RNA 5' pyrophosphohydrolase. By utilizing the construction method and the kit, nucleic acid information of various short RNA fragments can be obtained; and the construction method and the kit are applied to the field of life science.

The invention discloses an electrochemical analysis method for determining the activity of T4 polynucleotide kinase based on a magnetic nano material, the method comprises the following steps: preparing Fe3O4@TiO2 magnetic core-shell nanoparticles by a hydrothermal method, phosphorylating the 5'end of a nucleic acid chain S1 when ATP and T4 PNK exist, and modifying the surface of the Fe3O4@TiO2 magnetic nanoparticles with the end phosphorylated S1 by specific reaction with TiO2; adding a gold nanoparticle-nucleic acid chain S2 compound (AuNPs-S2), and forming a Fe3O4@TiO2-S1/S2-AuNPs compound due to a hybridization reaction between the nucleic acid chain S1 and the nucleic acid chain S2; using hexamino ruthenium [Ru(NH3)63+] is used as an electroactivity indicator, and realizing high-sensitivity, rapid and quantitative determination of the activity of T4PNK through magnetic enrichment and electrochemical signal enhancement on the surface of GME.

The invention provides a kit and a method for constructing a DNA library, the kit comprises a mixed enzyme solution, a mixed buffer solution, ligase and a ligation buffer solution, the mixed enzyme solution comprises endonuclease, T4 DNA polymerase, Klenow fragments, Taq DNA polymerase and T4 polynucleotide kinase, the mixed buffer solution and the ligation buffer solution comprise DTT and PEG 8000, the mixed enzyme solution and the mixed buffer solution are used for forming a first reaction system, the ligase and the ligation buffer solution are used for forming a second reaction system, in the first reaction system, the working concentration of the endonuclease is 0.01-0.05 U/mu L, the working concentration of the T4DNA polymerase is 0.04-0.1 U/mu L, the working concentration of the Klenow fragment is 0.005-0.02 U/mu L, the working concentration of the Taq DNA polymerase is 0.025-0.075 U/mu L, the working concentration of the T4 polynucleotide kinase is 0.1-0.5 U/mu L, the working concentration of the DTT is 5-10mM, and the working concentration of the PEG 8000 is 1-10%. According to the invention, the kit and the method for constructing the DNA library, which can improve the library construction efficiency, can be provided.

The invention relates to a method and a kit in the technical field of biology, and concretely relates to a next-generation sequencing library building kit and method for improving the library conversion rate. The kit and the method are particularly suitable for next-generation sequencing application of trace samples. The kit comprises: enzyme mixed solutions which comprises a first enzyme mixed solution, a second enzyme solution and a third enzyme mixed solution, wherein the first enzyme mixed solution is formed by mixing T4 polynucleotide kinase, T4 DNA polymerase, a Klenow fragment and Taq enzyme according to a ratio of 3: 1: 2: 1, the second enzyme solution is T4 DNA ligase, and the third enzyme mixed solution is a mixed solution of 2X QuarTaq HiFi HotStart; buffer solutions which comprise a first buffer solution corresponding to the first enzyme mixed solution and a second buffer solution corresponding to the second enzyme mixed solution, wherein the second buffer solution is formed by mixing Tris-HCl with the concentration of 0.1-0.6 M, MgCl2 with the concentration of 0.05-0.1 M, DTT with the concentration of 0.01-0.05 M, ATP with the concentration of 1-10 mM, PEG6000 with the concentration of 30%-50% and 1, 2 propylene glycol with the concentration of 10-20%; and a linker, an index sequence and a universal primer.

The invention provides a cfDNA (circulating cell free DNA) terminal repairing enzyme composition, which comprises the following enzymes, including an enzyme I and an enzyme II, wherein the enzyme I enables cfDNA to carry out DNA double-strand repairing and 3' terminal plus A when dNTP (deoxy-ribonucleoside triphosphate) is in the presence, and is a Taq DNA polymerase and Klenow Fragment, Exo-, andthe enzyme II enables a cfDNA 5' terminal to generate phosphorylation modification and is T4 Polynucleotide Kinase (T4 PNK for short). The invention also provides a cfDNA terminal repairing buffer solution reagent. The invention also provides a method which uses the above reagent to carry out cfDNA terminal repairing and construct a cfDNA sequencing library. The invention has the following technical effects that a use amount of reagent enzymes is small, cost is low, repairing efficiency is unexpectedly high, and therefore, the invention is suitable for carrying out terminal repairing on the cfDNA with a low sample amount.

The invention discloses an economical and practical nucleic acid chain 5'-hydroxyl phosphorylation method. The method comprises the following steps of taking purchased non-modified DNA (deoxyribonucleic acid) short fragments as raw materials, taking the 5'-end of the short fragments as hydroxyl, taking 10 times of a commercially purchased phosphorylation buffer solution component as a phosphoric acid source, and phosphorylating the 5'-hydroxyl of DNA under the action of T4 polynucleotide kinase. The method provided by the invention is wide in application range, mild in reaction condition, simple in post-treatment, high in recovery rate, low in cost and wide in application prospect.

The invention discloses a method for detecting pathogenic microorganisms. The method comprises the following steps: simultaneously separating and extracting DNA and RNA from a sample of a subject; performing reverse transcription on the extracted RNA to obtain cDNA; treating the extracted DNA and the extracted cDNA by using a composition containing a plurality of enzymes, so as to obtain treated DNA and treated cDNA; performing PCR amplification on the basis of the treated DNA and the treated cDNA so as to construct a sequencing library; performing metagenome sequencing on the basis of the sequencing library to obtain a metagenome sequencing result of the sample; and determining the pathogenic microorganisms in the sample based on the metagenome sequencing result, a pathogenic microorganism genome database and a species classification database. The invention also discloses a kit for establishing the microbial metagenome sequencing library. The kit comprises a fragmentation and terminal repair system, wherein the fragmentation and terminal repair system comprises DNA incision enzymes, T4 polynucleotide kinase, T4 DNA polymerase, a Klinno fragment and Taq enzymes.

The invention relates to the field of analysis and detection, in particular to a detection model for non-labeled and non-fixed cathode photoelectrochemical detection of T4 polynucleotide kinase as well as a preparation method and application of the detection model. The construction method of the detection model comprises the following steps: (1) preparation of a Bi2O3 nano material; (2) preparation of a Bi2O3/ITO electrode; (3) preparation of biological reaction solutions with different T4 polynucleotide kinase concentrations; (4) determination of light current; when the detection model is used for detecting the T4 PNK, biomolecule immobilization is not needed, the operation is simple and convenient, the cost is low, the selectivity is good, the sensitivity is high, the linear range is wide (5.0 * 10 <-4 >-10 U/mL), the detection limit is low (1.0 * 10 <-4 > U/mL), and the detection model has huge potential in practical application.

The invention discloses a novel method for detecting the activity of T4 polynucleotide kinase (T4 PNK) in complex biological matrix samples with high salt, high protein and the like by using silver nanoclusters (ssDNA/AgNCs) with stable oligonucleotide sequences. A large amount of salt and protein exist in a complex biological matrix sample, and the complex biological matrix sample needs to be subjected to high-multiple dilution, otherwise, fluorescence detection of T4 PNK is difficult to realize. Researches find that when enzymatic reactions such as T4 PNK and the like are completed, firstly, a quantitative AgNO3 solution is added, secondly, NaIO4 and Zn (NO3)2 solutions are added to oxidize protein, finally, dark-state ssDNA/AgNCs is added into the solution, bright-state ssDNA/AgNCs can be obtained after incubation, and fluorescence detection of the activity of T4 PNK is carried out. The method has the advantages of high detection sensitivity and high anti-interference capability, and can be suitable for high-salt and high-protein complex biological matrix samples without dilution.

The invention discloses a nucleic acid molecule for coding T4 polynucleotide kinase, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO: 01, the invention further discloses a recombinant vector and a host cell containing the nucleotide sequence, an expression method of the recombinant vector and the host cell, and application of the produced T4 polynucleotide kinase. The nucleic acid molecule of the T4 polynucleotide kinase, disclosed by the invention, provides a technical guarantee for the yield, purity and activity of the T4 polynucleotide kinase.