58 results about "Klenow fragment" patented technology

Synthesis of the second strand of cDNA using pol ∝-primase is provided. Pol ∝-primase is used to initiate primers on a cDNA strand de novo, followed by extension of the primer with pol ∝-primase, a second DNA polymerase or a combination thereof. In a preferred embodiment, pol ∝-primase extends the primer, followed by addition of a second DNA polymerase, preferably Klenow fragment.

The invention discloses a high-throughput sequencing library and a construction method thereof. The construction method comprises the following steps: S1. acquiring a DNA fragment from a DNA-protein cross-linked complex; S2. enzymatically digesting the DNA fragment by virtue of exonuclease; and S3. constructing a library for the enzymatically digested DNA fragment so as to obtain the high-throughput sequencing library, wherein the exonuclease refers to nuclease which acts on double-stranded DNA only and has the activity of 3'->5' exonuclease rather than the activity of DNA polymerase. By replacing a klenow fragment which has the activity of the 3'->5' exonuclease and the activity of the DNA polymerase in the prior art by virtue of the exonuclease which has the activity of 3'->5' exonuclease and does not have the activity of DNA polymerase, the library construction method disclosed by the invention can be used for removing the marker in a non-target fragment more thoroughly, so that the purity of the obtained DNA fragment is greatly improved, and subsequently the effective quantity of subsequent sequencing data is increased.

The invention relates to compositions, methods, and kits for hot start polynucleotide synthesis, including extension of primed polynucleotide templates and polymerase chain reaction (PCR). Hot start is provided by a thermally inactivated blocking polymerase protein that binds primed polynucleotide templates and prevents their access to a thermostable nucleic acid polymerase. High temperatures employed in the synthesis reaction cause the blocking polymerase to denature, thereby permitting the action of a thermostable processive polymerase. Compositions of the invention include a specific blocking polymerase protein which is a mutant of the Klenow fragment of E. coli DNA polymerase. The mutant is essentially devoid of polymerase activity, processivity, and 3′ to 5′ exonuclease activity. Use of the thermally inactivated blocking polymerase together with a thermostable polymerase reduces non-specific priming and accumulation of unwanted amplification products, increasing the specificity and sensitivity of the synthesis reaction.

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 provides a DNA polymerase with DNA or RNA as a template. The DNA polymerase is obtained through amino acid replacement, including G198W, V222I, E306K, Q354E, A381E and E582K, of klenow fragments of escherichia coli polymerases I. The invention also provides a primer with a stem loop structure and used for constant-temperature nucleic acid amplification. The invention further providesa nucleic acid test technique and a test kit both based on combination between rapid constant-temperature nucleic acid amplification and a Cas test system. The DNA polymerase, the nucleic acid test technique and the test kit can be used for testing nucleic acid targets in test samples at a constant temperature, have the advantages of low cost, short test time, simplicity and convenience in operation, high specificity, high sensitivity and the like, and are particularly applied to POCT (point-of-care testing).

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 pertains to biotechnology and gene engineering fields, in particular to a gene cloning plasmid (pBCFX+ and pBCFX-) based on Phi BT1 integrase and Phi C31 integrase and the application thereof. The plasmid uses Phi BT1 integrase and tandem sequence of Phi C31fang integrase corresponding to attP site with interposition of LacZ expression frame as replacement cloning site of a plasmid vector (pBCFX+ and pBCFX-) with insertional inactivation which is used for doing blue-white screening. The cloning method based on the plasmid mainly comprises mixture of Phi BT1 integrase and Phi C31 integrase as site-directed integration kinase and additionally adding corresponding attB sequence to the two sides of inserting DNA fragment by using PCR, etc. according to the purpose to determine the direction of insertion fragment. The cloning system manipulates inserting DNA which contains special incision enzyme sites with convenience and has original predominance in manipulating Klenow fragment. The system simplifies the method of operation on recombinant DNA because of the omitting of manipulation of restricted enzyme, phosphatase and joinase and has extensive application prospect in gene engineering.

The invention discloses a microRNA detection method based on Hg<2+> assistance and a double-polymerase isothermal nucleic acid amplification technology, and belongs to the technical field of optical bio-sensing. On the basis of a combined catalytic action of a polymerase Klenow fragment and terminal deoxynucleotidyl transferase, microRNA is effectively extended into a polythymine base sequence which is relatively long; Hg<2+> is added, so that a T-Hg<2+>-T double-stranded structure is formed; an SYBR Green I fluorescent dye is interpolated into the T-Hg<2+>-T double-stranded structure, so that the fluorescence of the SYBR Green I is enhanced; and a fluorescence enhancing degree is positively related to the concentration of the microRNA; therefore, the sensitive detection of the microRNA is achieved.

The invention discloses an image visualization method for detecting single molecule DNA duplication. The method comprises the steps that single-chain DNA and a buffering system of a DNA polymerase Klenow fragment combined with the single-chain DNA are scanned with an atomic force microscope, and DNA duplication is subjected to imaging observation through the atomic force microscope, wherein the single-chain DNA contains dNTP, and the two ends of the single-chain DNA are fixed to two different positions on hollow DNA folded paper respectively. According to the method, the DNA duplication process is tracked on the single molecule level, the whole process of combination of the single-chain DNA and single DNA polymerase, moving between the DNA chain and the DNA polymerase and conversion from the single-chain DNA chain to a double-chain DNA chain is recorded, various forms of state distribution of the DNA chain on the surface of a substrate and the changing situation in the interaction process of DNA and the DNA polymerase can be captured, the biomolecule behavior action mechanism can be understood more easily, and data on the time scale and the space scale in the molecule event obtaining process can be obtained more easily.

The invention relates to a constant temperature reaction-based method for detecting microRNA in a solution to be detected. The method comprises that an electrode is immersed in a first DNA fragment molecular solution, undergoes a reaction at a temperature of 37 DEG C for 6h, then is immersed in a mercapto hexanol solution, undergoes a reaction for 1h, then is immersed in a silver nanoparticle solution, undergoes a reaction for 1h, then is immersed in a second DNA fragment molecular solution and undergoes a reaction for 0.5h, a microRNA standard liquid is prepared, the electrode is immersed in the microRNA standard liquid, a third DNA fragment molecule, a Klenow fragment and dNTPs are added into the microRNA standard liquid, the electrode undergoes a reaction in the mixed liquid at a temperature of 37 DEG C for 1h, then is immersed in a Nt. BbvCI solution and undergoes a reaction for 1h, wherein in the detection, a linear scanning voltammetry method is used, a standard curve graph is drawn, an electrical signal of the liquid to be detected is acquired by the above processes, and the electrical signal and the standard curve graph are compared so that a microRNA concentration of the liquid to be detected is obtained. The constant temperature reaction-based method has the advantages of simple processes, high sensitivity, good specificity, low background signal and wide application range.

The invention discloses a method for performing 3' end biotin labeling on DNA fragments obtained through ultrasonication. The method comprises the following steps of: I, adding biotin-labeled dCTP while treating the DNA fragments by Klenow fragments and/or T4DNA polymerase, and then filling in; II, adding biotin-labeled dATP while treating DNA fragments by Klenow fragments and/or T4DNA polymerase, and then filling in; III, adding biotin-labeled dGTP while treating DNA fragments by Klenow fragments and/or T4DNA polymerase, and then filling in; and IV, adding biotin-labeled dTTP while treating DNA fragments by Klenow fragments and/or T4DNA polymerase, and then filling in. The method has the advantages that the labeling efficiency is high; the application range is wide; and the steps of the method are few, and thus the method is simple, convenient and feasible.

The invention discloses a screening method of molecular reverse probes for capturing a specific region of an enriched genome. The method comprises the steps that firstly the structures of the molecular reverse probes are designed; starting from the 5' end of the target region, the probes with at least 50% of scanned areas falling on the 5' end are selected to serve as a candidate starting set; adjacent probes are scored in pairs, the probes meeting the requirement are combined with the previous selected probes sequentially to form a set of all possible probe combinations, each candidate combination is scored, score calculation is conducted on all possible combinations, and the combination with highest scores or lowest penalty scores is obtained. The method is applicable to linker structures of different sequencing platforms, an obtained molecular reverse probe technique is remarkably improved at the aspects of precision, convenience, detection sensitivity and the like, meanwhile, a coverage target region, especially the number of the probes needed by a klenow fragment exon region is effectively reduced, and the method is convenient and rapid, and saves the cost.

The invention discloses a detection method of microRNA (micro Ribonucleic Acid), and belongs to the field of optical biosensing techniques. A to-be-detected sample is mixed with template DNA (Deoxyribonucleic Acid), an obtained first mixture is annealed and hybridized, a buffer solution containing polymerase Klenow fragment, deoxyuracil nucleoside triphosphate and terminal deoxynucleotidyl transferase is added into a reaction product, an obtained second mixture is caused to react for 4h at 35 DEG C in a water bath, so as to prepare a polyuracil base sequence; sodium ascorbate is added into a polyuracil base sequence solution obtained in the step (4), and then CuCl2 is added into an obtained third mixture, and an obtained fourth mixture is caused to react for 10min at a room temperature, so as to prepare a red-fluorescence copper nano cluster which uses the polyuracil base sequence as a template, thereby obtaining a solution containing the red-fluorescence copper nano cluster. The intensity of the red fluorescence of the solution including the red-fluorescence copper nano cluster is measured by adopting a spectrofluorophotometer; by comparing the result about the intensity of the red fluorescence with a standard relation curve, the concentration of the microRNA is calculated and obtained.

The invention discloses a method for detecting influence of mechanical force on interaction of DNA (deoxyribose nucleic acid) and DNA polymerase. The method comprises steps as follows: a single-stranded DNA template containing dNTP (deoxyribonucleotide triphosphate) and a buffering system of a DNA polymerase Klenow fragment combined with the single-stranded DNA template are scanned by means of an atomic force microscope, two ends of the single-stranded DNA template are fixed on two different sites on inner edges of hollow DNA origami, the two different sites are not located on the edge of the same straight line, the buffering system is arranged on a newly dissociated mica substrate, and synthesis of double-stranded DNA is observed through imaging of the atomic force microscope. The information about the effect of mechanical force in the non-double-helix direction on the structure and the function of DNA and DNA polymerase is acquired with the detection method, then influence of the mechanical force on DNA synthesis is obtained, accurate positioning on the nano scale can be realized, and a condition is provided for detection of the interaction relation of DNA and DNA polymerase under a certain tension condition is provided.

The invention discloses an amplification primer and an amplification method of a complete mitochondrial genome sequence of nibea miichthioides chu. The amplification primer comprises 4 klenow fragment amplification primer pairs and 19 genome walking primers. According to the amplification primer and the amplification method provided by the invention, the complete mitochondrial genome sequence of nibea miichthioides chu, which is obtained by conducting PCR amplification on genome of the nibea miichthioides chu, can provide a material base for conservation genetics and evolutionary genetics of the nibea miichthioides chu and for marker-assisted selection of high-quality breeding varieties.

The invention discloses a plant-anthocyanin synthesis control gene and application thereof. The sequence of the disclosed gene is shown in the SEQ ID No.1. A 3,772-bp klenow-fragment insertion mutation of the gene exists in first intron of white heading brassica rapa, and the gDNA sequence of the mutation is shown in the SEQ ID No.5. According to the mutation in the intron in the gDNA nucleotide sequence of the gene, three specificity PCR primers of the Marker 1, the Marker 2 and the Marker 8 are designed, wherein the Marker 1 and the Marker 2 are co-dominant markers, and the Marker 8 is a maternal marker. The DNA of the target brassica rapa is subjected to PCR amplification and identified through agarose gel electrophoresis, drinamyl hybrid strains and homozygous strains in segregation population can be rapidly identified through the Marker 1 and the Marker 2, and the drinamyl hybrid strains and the homozygous strains in the segregation population can also be distinguished through theMarker 8 in cooperation with the heading characteristics.

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 discloses a biosensor for detecting tobramycin. The biosensor comprises an aptamer probe, a hairpin probe H1, a hairpin probe H2, Klenow Fragment polymerase, Nt.BbvCI endonuclease, a buffer solution and graphene oxide. The sequence of the aptamer probe is as shown in SEQ ID No: 1, the aptamer probe is formed by hybridizing an aptamer Aptamer with the sequence as shown in SEQ ID No: 2 and an amplification template T with the sequence as shown in SEQ ID No: 3. The sequence of the hairpin probe H1 is as shown in SEQ ID No: 4, and the sequence of the hairpin probe H2 is as shown in SEQ ID No: 5. When the biosensor is used for detecting tobramycin, the method is simple, the practicability is good, the stability is high, the detection lower limit reaches 0.06 nM and is lower than that of the existing similar sensors, and the biosensor has wide application prospects in the fields of environmental monitoring and food safety.