116 results about "Taq polymerase" patented technology

Disclosed are mutant DNA polymerases having improved extension rates relative to a corresponding, unmodified polymerase. The mutant polymerases are useful in a variety of disclosed primer extension methods. The mutant polymerases overcome the inhibitory effects by an intercalating dye. Therefore, the mutant polymerases are useful in a variety of disclosed methods in combination with an intercalating dye. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the mutant DNA polymerases.

The present invention provides mutants of DNA polymerases having an increased rate of incorporation of nucleotides into nucleic acids undergoing polymerization and having an enhanced resistance to inhibitors of DNA polymerase activity. The mutant polymerases are well suited for fast PCR applications, for PCR amplification of targets in samples that contain inhibitors of wild-type polymerases, and for fast PCR amplification of samples containing DNA polymerase inhibitors. In exemplary embodiments, the mutants are mutants of Taq DNA polymerase.

Formulations for dry storage of PCR reagents are described. These formulations find use in manufacture of self-contained microfluidic card devices for PCR clinical testing in which the reagents are reconstituted at the point of testing. In these cards, TAQ polymerase is stored “on-board” in vitrified dry form without lyophilization or freezing, and is reconstituted by either the sample or a sample eluate during the assay.

The invention discloses a mutant Taq DNA (deoxyribonucleic acid) polymerase. Amino acid sequences of a wild Taq DNA polymerase are mutated, particularly, 605th leucine of the wild Taq DNA polymerase is mutated to obtain arginine, 617th arginine of the wild Taq DNA polymerase is mutated to obtain phenylalanine, 667th phenylalanine of the wild Taq DNA polymerase is mutated to obtain leucine, and 480th-485th amino acid of thumb zones of the Taq DNA polymerase is replaced by TBD zones, namely, 262nd-337th amino acid of T3 DNA polymerase. The mutant Taq DNA polymerase has the advantages that the activity, the tolerance and the like of the mutant Taq DNA polymerase can be obviously improved owing to site-directed mutation; the mutant Taq DNA polymerase with the high enzyme activity and the high tolerance can be used in direct amplification PCR (polymerase chain reaction) systems without nucleic acid extraction. The invention further discloses a method for preparing the mutant Taq DNA polymerase and application thereof.

The invention provides a sequencing primer for qualitative detection of cytochrome oxidase CYP2C19 genetic typing and a kit of the sequencing primer and belongs to the field of external nucleic acid detection. The kit comprises uracil DNA (Deoxyribonucleic Acid) glycosylase, Taq polymerase, PCR (Polymerase Chain Reaction) reaction liquor, PCR amplification primers, pyrophosphoric acid sequencing primers and positive control products. The kit is high in sensitivity and good in specificity, PCR products can be simply treated for a pyrophosphoric acid sequenator for sequencing, operation is simple, reaction time is short, the sensitivity of the kit is higher than that of a golden standard-capillary electrophoresis sequencing, and the kit is suitable for use for mutation analysis.

Formulations for dry storage of PCR reagents are described. These formulations find use in manufacture of self-contained microfluidic card devices for PCR clinical testing in which the reagents are reconstituted at the point of testing. In these cards, TAQ polymerase is stored “on-board” in vitrified dry form without lyophilization or freezing, and is reconstituted by either the sample or a sample eluate during the assay.

The invention discloses an animal epidemic disease three-color fluorescence RT-PCR detection kit and a detection method thereof, and is characterized in that the kit comprises multiple fluorescence RT-PCR reaction mother liquor, a positive control, a negative control, an AMV reverse transcriptase, and a Taq polymerase, wherein the multiple fluorescence RT-PCR reaction mother liquor contains a multiple 10*fluorescence RT-PCR reaction buffer, an avian influenza primer and a probe, a newcastle disease virus primer and a probe, an avian infectious bronchitis primer and a probe, and bovine serum albumin, wherein sequences of the forward primers, reverse primers, and specific probes are as shown in SEQIDNO. 1, NO. 2, NO.3, NO.4, NO.5, NO.6, NO.7. NO.8, NO.9, and NO.10. The advantages of the invention are that avian influenza, newcastle disease, and avian infectious bronchitis viruses can be detected simultaneously in a same reaction tube; the specificity is strong; the sensitivity is high, is rapid and accurate.

The invention provides a phytophthora sojae molecule detecting primer and a detection reagent kit thereof. The primer comprises a forward primer PSD11 and a backward primer PSD12; and the detection reagent kit comprises PCR buffer solution, MgCl2, dNTPs, Taq polymerase, positive control DNA, DMSO, PSD11 / PSD12 and ultrapure water. The detection reagent kit based on the primer specifically amplifies specific amplifying products with the fragment length of 382bp in phytophthora sojae pure DNA, bacteria-bearing pathological tissues and soil. The specific molecule detecting primer and the detection reagent kit thereof can be applied to the rapid, sensitive and specific detection for plant tissues infected with phytophthora sojae and the phytophthora sojae in soil and can be also applied to the early diagnosis of field diseases and the high-sensitive and rapid molecule detection and identification for the phytophthora sojae carried by soybean exporting or importing from customs.

The invention discloses a mixed type hot-start DNA polymerase composition, a PCR amplification kit, and applications thereof. The composition comprises Taq DNA polymerase, an anti-Taq antibody, and mutant type KOD DNA polymerase. The anti-Taq antibody is capable of specifically being combined with Taq DNA polymerase. The 147th site of wild type KOD DNA polymerase is histidine, and the 147th site of mutant type KOD DNA polymerase is lysine. The experiment results show that the mismatch rate is low when the mixed type hot-start DNA polymerase composition is used for PCR amplification, the composition has the dual characteristics of high efficient amplification and high fidelity and is heat resistant in amplification, and the amplification effect is good no matter for a long DNA segment, a template with a low amount, or a template with a high GC content.

The invention provides a human ALDH2 genotype detection kit comprising a PCR amplification primer, DNA polymerase and a reaction buffer solution; wherein the DNA polymerase is a polyethylene glycol (PEG) modified hot-start Taq polymerase which can tolerate the PCR reaction inhibitors in blood; and further provides the nucleotide sequence of the PCR amplification premier. The kit has the following advantages: (1) the DNA polymerase is a polyethylene glycol (PEG) modified hot-start Taq polymerase which can tolerate the PCR reaction inhibitors in blood, thus can realize the human whole-blood PCR amplification without carrying out nucleic acid extraction, and is capable of directly carrying out PCR amplification in blood; (2) the kit can carry out detection on a sample ALDH2 genotype.

The invention relates to the use of enzymes for copying or amplifying bisulphite modified or treated nucleic acids, wherein the enzymes are more effective in copying or amplifying the nucleic acid compared with native Taq polymerase under substantially the same conditions.

The invention belongs to the technical field of biology, and relates to a PCR (Polymerase Chain Reaction) experiment. Particularly, the invention provides a PCR premixed solution, which comprises Taq polymerase, a buffer solution, dNTP, glycerin, sodium azide and 1, 2, 4-triazole and the like. The PCR premixed solution provided by the invention can stably exist for more than 144 hours at normal temperature; the work efficiency is improved; and the cost is reduced.

The invention provides a Taq DNA polymerase mutant and application thereof. The Taq DNA polymerase mutant is an amino acid sequence formed by insertion, substitution or depletion of one or more aminoacids in an amino acid sequence of Taq DNA polymerase shown as SEQ ID NO. 1, or by addition or deletion of one or more amino acids in the sequence of SEQ ID NO. 1. Compared with Taq DNA polymerase shown as SEQ ID NO. 1, the amino acid sequence has enhanced amplification sensitivity and increased yield. The Taq DNA polymerase mutant has higher sensitivity and yield than original Taq DNA polymeraseand is also suitable for multiple amplification of low-quality samples.

The invention provides an Oncidium EST-SSR labeling primer and application thereof. The primer comprises a primer pair as follows: 5'-ATCGTAATCCTGAAGCGTATC-3' and 5'-AAGCCCAAACTATTCCATT-3'. The application of the Oncidium EST-SSR labeling primer comprises the steps of carrying out PCR amplification by taking a DNA sample extracted from Oncidium plants as a template, and labeling Oncidium molecules, wherein a reaction system includes the following components by volume (20mu L in all): 11.3mu L of ddH2O, 2mu L of 10*Buffer, 2mu L of 2.5mmol.L<-1> dNTPs, 1 microliter of each of 10mu mol.L<-1> primers in the primer pair, 0.2mu L of 5U.mu L<-1> Taq polymerase and 2.5mu L of 20ng / mu L template DNA.

The invention discloses a Taq DNA polymerase activity detection method comprising design of a template and a primer, preparation of a PCR reaction system, setting of PCR reaction conditions and judgment of activity of a Taq DNA polymerase. The method is easy and convenient to operate, accurate, high in sensitivity, and capable of effectively detecting weak activity of the Taq DNA polymerase. The method solves the problem that an end-point method is multiple in steps, long in time consuming and large in influence caused by human factors, and also solves the problem that accuracy of detection of the activity of Taq enzyme is affected by the change of activity of the Taq enzyme. The Taq DNA polymerase activity detection method disclosed by the invention establishes a detection standard of activity of the Taq DNA polymerase, overcomes the subjectivity and randomness of methods in prior art, and is objective and efficient. The method can be used for promoting the development of related technologies of the Taq DNA polymerase and driving the improvement of PCR related technologies, and has relatively strong practicability. The invention can not only be used for the detection of the activity of the Taq DNA polymerase, but also be used for the detection of the activity of other heat-resistant DNA polymerases, such as Tth DNA polymerases, and pfu DNA polymerases.

The invention relates to a sequencing primer pair for detecting an ALDH2 (Aldehyde Dehydrogenase 2) genotype with a pyrosequencing method and a kit thereof, and belongs to the technical field of in-vitro nucleic acid detection. The primer pair comprises a positive amplification primer, a reverse amplification primer and a sequencing primer, wherein a 5' end of the positive amplifier primer is subjected to biotin labeling. The kit comprises the positive amplification primer, PCR (Polymerase Chain Reaction) reaction liquid containing the reverse amplification primer, the sequencing primer, uracil DNA (Deoxyribose Nucleic Acid) glycosylase and Taq polymerase. The kit provided by the invention has the advantages of accurate detection result, high specificity, short detection period, easiness in operation, and capability of effectively meeting clinical examination requirements. Moreover, the kit further has the advantages that a reaction process can be monitored in real time, the reaction time is short, a PCR product can be subjected to pyrosequencing by a pyrosequencing instrument and high-flux sample detection through simple treatment, and the sensitivity is higher compared with a golden standard method, namely, a capillary electrophoresis sequencing method.

The invention discloses a detection primer and a detection kit for watermelon wilt disease fungi. The detection primer is a dual-detection primer set consisting of a wilt disease fungus universal primer and a primer which only can amplify a watermelon wilt disease fungi. A reagent of the detection kit comprises the detection primer, a buffer solution, dNTPs, Taq polymerase, watermelon wilt disease fungus positive control DNA and ultrapure water. The invention further discloses a detection method. The detection method comprises the following steps: (1) extracting DNA of a plant tissue infected by the watermelon wilt disease fungi; (2) carrying out PCR amplification on DNA which is separated in the step (1) by virtue of the detection kit; and (3) separating PCR amplification products in the step (2) by virtue of sepharose gel electrophoresis, dyeing the products with ethidium bromide under an ultraviolet lamp after the separation, and determining a result according to the sizes of the amplified products. According to the invention, a method for detecting watermelon wilt disease fungi at a biological molecular level is provided and has the characteristics of high precision and convenient operation.

The triplex PCR-based rapid detection kit for Streptococcus suis Type 2 which is composed of four cryopreservation tubes containing different components, 100 PCR reaction tubes and the brochure of the detection steps for the cryopreservation tubes. The four cryopreservation tubes contain mixture of 10*buffer, dNTP, primer gdh-1, gdh-2, mrp-1, mrp-2,cps2J-1, cps2J-2 and Taq DNA polymerase; lysis buffer; positive control solution; diluent respectively. The triplex PCR process established in the present invention can amplify gene gdh, mrp and cps2J of SS-2 at the same time and has high specificity. The homology of sequencing is always between 98.84% and 100% exhibiting high accuracy and specificity.

The invention provides a human HLA-B27 gene typing kit. The human HLA-B27 gene typing kit comprises a PCR amplification primer, DNA polymerase and a reaction buffer solution, wherein the DNA polymerase is a hot initiated Taq polymerase which is modified by polyethylene glycol (PEG) and can tolerate PCR reaction inhibitors in blood. The invention also provides a nucleotide sequence of the PCR amplification primer. The human HLA-B27 gene typing kit has the technical effects that the adopted DNA polymerase is the hot initiated Taq polymerase which is modified by PEG and can tolerate PCR reaction inhibitors, PCR amplification on whole blood of human (nucleic acid extraction does not need to be carried out) can be realized, PCR amplification is directly carried out by virtue of blood, and DNA extraction does not need to be carried out; the human HLA-B27 gene typing kit can realize HLA-B27 negative and positive identification on a sample; meanwhile, a gene subtype result of an HLA-B27 positive sample can be obtained, and 24 HLA-B27 gene subtypes and other gene subtypes of 13 HLAB sites can be detected by every eight holes (eight linked pipes).

The invention discloses a double-temperature multiple RT-PCR kit for simultaneously detecting two orchid viruses and a preparation method thereof, relates to a plant virus detecting kit, and provides a double-temperature multiple RT-PCR kit capable of simultaneously detecting two orchid viruses in one detection process and an application method thereof. The kit comprises a reagent A, a reagent B, a reagent C, a reagent D and a reagent E, wherein the reagent A is an RT reaction liquid which contains an M-MuLV reverse transcriptase buffer solution, random primers and dNTP; the reagent B is an M-MuLV reverse transcriptase; the reagent C is a PCR reaction liquid which contains a PCR buffer solution, MgCl2, dNTP and detection primers; the reagent D is a Taq polymerase; and the reagent E is a reference substance. Both positive control and negative control are established for each detection, and the application method comprises the following steps: carrying out reverse transcription; carrying out amplification detection, and determining the sizes of amplified bands according to the conventional agarose gel electrophoresis by taking 5 mu L of reaction products. The invention is especially applicable to the use by orchid enterprises, port quarantine authorities and related scientific research institutions.

The invention relates to a Taq DNA polymerase, and PCR (polymerase chain reaction) fluid and application thereof. Compared with SEQ ID NO:1, the amino acid sequence of the Taq DNA polymerase has following mutations: p.E641K, p.Q698E, and p.E708Q or p.E708D. By the Taq DNA polymerase, blood resistance and PCR inhibitor are improved obviously, and blood samples and soil samples can be directly used for PCR detection. Therefore, experimental time and cost can be greatly saved for experimenters, inter-sample cross contamination caused by multi-step operation can be avoided, and PCR detection results are more credible; an alternative way is provided for PCR detection of precious micro samples.

The present invention describes methods for the production and use of single chain (single-stranded) fluorescence resonance energy transfer (“FRET”) DNA or RNA aptamers containing fluorophores (F) and quenchers (Q) at various loci within their structures, such that when its specific matching analyte is bound and the FRET-aptamers are excited by specific wavelengths of light, the fluorescence intensity of the system is modulated (increased or decreased) in proportion to the amount of analyte added. F and Q are covalently linked to nucleotide triphosphates (NTPs), which are incorporated by various nucleic acid polymerases such as Taq polymerase during the polymerase chain reaction (PCR) and then selected by affinity chromatographic, size-exclusion or molecular sieving, and fluorescence techniques. Further separation of related FRET-aptamers can be achieved by ion-pair reverse phase high performance liquid chromatography (HPLC) or other types of chromatography. Finally, FRET-aptamer structures and the specific locations of F and Q within FRET-aptamer structures are determined by digestion with exonucleases and mass spectral nucleotide sequencing analysis.

The invention relates to a primer pair for detecting CYP2C9 genetic typing through a pyrosequencing method and a kit, and belongs to the technical field of in vitro nucleic acid detection. The primer pair comprises a CYP2C9*2 forward amplification primer, a CYP2C9*2 reverse amplification primer, a CYP2C9*2 sequencing primer, a CYP2C9*3 forward amplification primer, a CYP2C9*3 reverse amplification primer and a CYP2C9*3 sequencing primer. Biotin labeling is conducted at the 5' end of the CYP2C9*2 forward amplification primer and the 5' end of the CYP2C9*3 reverse amplification primer. The kit comprises the amplification primers, a PCR reaction solution 1, a PCR reaction solution 2, the sequencing primers, uracil DNA glycosylase and Taq polymerase. The kit has the advantages of being accurate in detection result, high in specificity, short in detection period, easy to operate and capable of effectively meeting the clinical examination requirement.

The invention provides a sequencing primer for detecting mutation of codons 12 and 13 in a second exon and a codon 61 in a third exon of a KRAS gene, and a kit thereof, and belongs to the field of in vitro nucleic acid detection. The kit comprises uracil DNA glycosylase, Taq polymerase, PCR amplification primers and sequencing primers. The kit provided by the invention has high sensitivity and good specificity; the PCR products can be simply treated for sequencing on a phosphate sequenator; and the kit has advantages of simple operation, short reaction time, and higher sensitivity than a gold standard-capillary electrophoresis sequencing, and is more suitable for mutation analysis.

The invention provides a fluorescent PCR kit for qualitative detection of HLA-B*1502 gene subtypes, and belongs to the field of in-vitro nucleic acid detection. The kit comprises uracil DNA glycosylase, Taq polymerase, PCR genotyping primers and a fluorescence probe; the PCR genotyping primer sequence is as shown in SEQ ID NO: 3-4 and / or SEQ ID NO: 6-7. The kit provided in the invention has high sensitivity and good specificity, and can monitor reaction process in real time and the reaction time is short; in addition, closed tube operation is performed, and subsequent treatment is not needed, which can maximally avoid the pollution of a reaction product, thus being capable of replacing traditional cell detection.

The invention discloses a nested PCR (polymerase chain reaction) kit for detecting duck-manure pollution in a water body and a detection method thereof, and belongs to the field of the detection of pollution in water bodies. The nested PCR kit for detecting the duck-manure pollution in the water body comprises two pairs of nested PCR primers, dNTP (deoxyribonucleoside triphosphate), a PCR buffer, a Taq polymerase, a Mg<2+> solution and ddH2O (double distilled water). Two rounds of PCR amplification are used by a nested PCR method. The detection method comprises the following steps of extracting a DNA (deoxyribonucleic acid) in a to-be-detected water sample, and carrying out a first round of PCR amplification by using the DNA in the to-be-detected water sample as a template, wherein the primers are an SDF (stroma derived factor) and SDR (short-chain dehydrogenase / reductase); after a first reaction is terminated, carrying out a second round of PCR amplification by using a product of the first round of PCR amplification as a template, wherein the primers are an NDF (neutral detergent fiber)and NDR, and after a second reaction is terminated, detecting the existence of a 158bp strip, which shows that the water body is subjected to the duck-manure pollution, through agarose gel electrophoresis. According to the method, the detection sensitivity is greatly increased through nested PCR amplification; a little duck-manure pollution which exists in water can be detected; moreover, the method has a favorable specificity, and can be directly applied to the detection and the preventive treatment work of fecal pollution in the water body.

The invention provides a sequencing primer for qualitative detection of TPMT genetic typing and a kit thereof, and belongs to the field of in vitro nucleic acid detection. The kit comprises uracil DNA glycosylase, Taq polymerase, a PCR reaction liquid, PCR primers, pyrosequencing primers and a reference substance. The kit provided by the invention has high sensitivity and good specificity; the PCR products can be simply treated for sequencing on a phosphate sequenator; and the kit has advantages of simple operation, short reaction time, and higher sensitivity than a gold standard-capillary electrophoresis sequencing, and is more suitable for SNP analysis.

The invention relates to a primer pair and kit for detecting VKORC1 (vitamin K epoxide reductase complex subunit 1) genotyping by pyrosequencing, belonging to the technical field of in vitro nucleic acid detection. The primer pair comprises VKORC1 G1639A and VKORC1 C1173T forward amplification primers, VKORC1 G1639A and VKORC1 C1173T reverse amplification primers and VKORC1 G1639A and VKORC1 C1173T sequencing primers, wherein 5' terminals of the VKORC1 G1639A forward amplification primer and the VKORC1 C1173T reverse amplification primer are respectively subjected to biotin labelling. The kit comprises the amplification primers, a PCR (polymerase chain reaction) liquid 1, a PCR liquid 2, the sequencing primers, uracil DNA (deoxyribonucleic acid)glycosylase and Taq polymerase. The kit provided by the invention has the advantages of accurate detection results, high specificity, short detection period, simplicity in operation, capability of effectively meeting the requirements of clinical examination, capability of monitoring the reaction process in real time, short reaction time, sequencing of PCR products on a pyrosequencing instrument after the PCR products are simply treated, high throughput sample detection and higher sensitivity than gold standard methods, namely capillary electrophoresis sequencing methods.

The invention relates to a primer pair and kit for detecting CYP3A4 genotyping by pyrosequencing, belonging to the technical field of in vitro nucleic acid detection. The primer pair comprises a forward amplification primer, a reverse amplification primer and a sequencing primer, wherein a 5' terminal of the forward amplification primer is subjected to biotin labelling. The kit comprises the forward amplification primer, a PCR (polymerase chain reaction) liquid containing the reverse amplification primer, the sequencing primer, uracil DNA (deoxyribonucleic acid)glycosylase and Taq polymerase. The kit provided by the invention has the advantages of accurate detection results, high specificity, short detection period, simplicity in operation, capability of effectively meeting the requirements of clinical examination, capability of monitoring the reaction process in real time, short reaction time, sequencing of PCR products on a pyrosequencing instrument after the PCR products are simply treated, high throughput sample detection and higher sensitivity than gold standard methods, namely capillary electrophoresis sequencing methods.