67 results about "Deoxynucleoside triphosphate" patented technology

A novel limited primer extension reaction improves detection sensitivity and specificity in a variety of hybridization platforms. In the invention, a sequence of target DNA that lacks one of the four types of nucleic acid bases for a span of eight or more adjacent nucleotide positions is selected for use. This sequence is referred to as the extension complement sequence, or ECS. A primer with a sequence that is complementary to the target sequence that is immediately downstream (to the 3′ side) of this ECS is used to initiate an extension reaction. Extension occurs using a DNA polymerase and standard deoxynucleoside triphosphates for three of the four types of nucleic acid bases. The fourth base, which is complementary to the base missing in the ECS, is either absent or present only in the form of a dideoxynucleoside triphosphate, which does not support further extension. In either case, the extension reaction does not proceed past the first occurrence in the template of the base that is missing in the ECS. This results in a primer extension product with fixed length determined by the length of the ECS. The process can be repeated using a thermal-stable polymerase in a thermal-cycled reaction that results in a linear amplification of the targeted sequence. The resulting limited primer extension products serve as ideal hybridization analytes for determination of sample sequence content using microarrays.

An object to be achieved by the present invention is to provide a nucleic acid amplification method by which a nucleic acid can be amplified using oligonucleotide primers and DNA polymerase. The present invention provides a nucleic acid amplification method which comprises performing incubation of a reaction solution containing at least one type of deoxynucleotide triphosphate, at least one type of DNA polymerase, at least two types of oligonucleotide primer, and the nucleic acid fragment as a template so as to perform a polymerase reaction that initiates from the 3′ end of the primer and thus amplifying the nucleic acid fragment, wherein a tag sequence is added at the 5′ end of the first oligonucleotide primer, and the tag sequence is a nucleotide sequence on the template nucleic acid fragment which is present downstream of the sequence which is substantially complementary with the 3′ end region of the first oligonucleotide primer (a region where the first oligonucleotide is annealed to the template nucleic acid).

This invention provides methods for pyrosequencing and compositions comprising 3′-O— modified deoxynucleoside triphosphates.

A method of amplifying RNA template is provided. The method comprises reverse-transcribing a ribonucleic acid (RNA) template to form a cDNA using a first reaction mixture comprising RNA template, at least one primer capable of hybridizing to the RNA template, a reverse transcriptase and deoxynucleoside triphosphates (dNTPs); and amplifying the cDNA to form an amplified product using a second reaction mixture comprising at least one strand displacement DNA polymerase, at least one inosine-containing primer and a nuclease that is capable of nicking DNA 3' to an inosine residue of the primer. The method is accomplished under an isothermal condition without denaturing the cDNA template. A method of quantifying RNA template in a sample and a method of detecting RNA template in a sample are also provided. Kits for amplifying deoxynucleic acid (DNA) from ribonucleic acid (RNA) template are also provided.

Aspects of the invention provide novel and surprisingly effective methods for the detection of nucleic acids, comprising nucleic acid amplification using base-modified deoxynucleoside 5′-triphosphates (dNTPs). Particular aspects relate to methods for enhancing hybridization properties of oligonucleotide primers and probes in assays detecting nucleic acids, comprise amplifying target DNAs in presence of base-modified duplex-stabilizing deoxyribonucleoside 5′-triphosphates to provide for modified target DNAs, and thereby considerably improving performance of the detection assays. The disclosed methods allow for increasing of the reaction temperature in PCR-based detection systems or, alternatively, reducing the length of the oligonucleotide primers and probes. Certain aspects relates to improvement of real time PCR assays, wherein nucleic acids of interest are detected as the reaction proceeds using fluorescent agents or oligonucleotide FRET probes.

The present invention relates to a novel set of four fluorescently labeled dye terminators. Two of them are single dye labeled terminators and the other two dye terminators are based on fluorescent resonance energy transfer (FRET). The FRET dye terminators are generated from the 4′,5′-bis-aminomethylfluorescein. Of the two amino groups of the donor dye, 4′,5′-bis-aminomethylfluorescein, one amino group is used to attach the acceptor dye, and the other amino group is used to attach the dideoxynucleoside-5′-triphosphate. Their preparation, energy transfer efficiency, and use as labels, specifically, in DNA sequencing reactions is disclosed.

The invention discloses a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for detecting single nucleotide polymorphism (SNP) of a Pax3 gene of yellow cattle. The method comprises the following steps: detecting gene polymorphisms according to DNA (deoxyribonucleic acid) pool sequencing results, wherein the gene polymorphisms comprise SNP of a sequence transcription start site -580 T or G of the Pax3 gene of the yellow cattle; in the presence of a Taq DNA polymerase, a Buffer (a buffer environment), Mg<++> and dNTPs (deoxynucleoside triphosphate), performing PCR (polymerase chain reaction) amplification on the Pax3 gene of the yellow cattle, digesting a PCR amplification product by HinfI and HaeIII restriction enzymes respectively, and performing agarose gel electrophoresis on enzyme digestion segments. According to the method disclosed by the invention, a foundation is laid for creating a relationship between the SNP and the growth trait of the Pax3 gene, so that the marker assisted selection of the growth trait of the yellow cattle in China can be conveniently carried out and a new strain of high-quality special beef in China can be quickly, efficiently and accurately bred.

The invention provides a dual fluorescence quantification RT-PCR (reverse transcription-polymerase chain reaction) detection kit, comprising a deoxynucleoside triphosphate mixture, MgCl2, an RNA enzyme inhibitor, a Moloney murine leukemia virus reverse transcriptase, a DNA polymerase, a influenza virus standard and a reference substance. Based on sequence analysis of present pervasive A H1N1 influenza virus, the invention provides a multiple fluorescence quantification PCR molecular biology gene diagnosis method and a diagnostic kit which are rapid, specific, accurate and sensitive. In addition, one reaction tube can simultaneously detect and differentiate an influenza A virus or an influenza B virus in 2h, so as to improve influenza detection.

The present invention provides a nucleic acid amplification method by using a oligonucleotide primer and a DNA polyase. The nucleic acid amplification method provided by the invention comprises performing incubation of a reaction solution containing at least one type of deoxynucleotide triphosphate, at least one type of DNA polymerase having strand displacement activity, at least two types of oligonucleotide primer, and the nucleic acid fragment as a template so as to perform a polymerase reaction that initiates from the 3' end of the primer and thus amplifying the nucleic acid fragment, wherein the characteristic is that a first oligonucleotide primer and a second oligonucleotide primer are designed in such a way that a region which contains two identical sequences X of serial 4 or more nucleotides within the region of 200 or less nucleotides, or a part thereof can be amplified.

The invention discloses a RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection kit and a RT-LAMP detection method for SVCV (spring viremia of carp virus). The RT-LAMP detection kit of SVCV comprises 10*ThermoPol Reaction buffer, Bst DNA (deoxyribonucleic acid) polymerase, dNTPs (deoxynucleoside triphosphates), AMV reverse transcriptase, outer primer F3 and B3, inner primer FIP, BIP and Betaine, MgC 12, DTT, and 1000*SYBR Green I. The RT-LAMP detection kit is simple, fast, and high in specificity and sensitivity, only needs a water bath kettle or a metal bath to accurately detect SVCV in samples in 2 hours, can detect SVCV of infected tissues of sick fish, can detect cells infected by SVCV, and is suitable for fast field detection of SVCV.

The present invention is directed to a method and a composition for amplifying and detecting a target nucleic comprising subjecting said target nucleic acid to a real time PCR amplification reaction in the presence of a thermostable DNA polymerase, a thermostable double strand dependent 3′-5′ exonuclease having a temperature optimum above 37° C., a pair of amplification primers, deoxynucleoside triphosphates, a detecting oligonucleotide carrying a first label and a second label, said first label being capable of acting as a fluorescent reporter entity when excited with light of an appropriate wavelength, said second label being capable of acting as a fluorescence quenching entity of said fluorescent reporter entity, characterized in that one label is bound to the 3′ end of said detecting oligonucleotide, and further characterized in that the other label is bound either internally or at the 5′ end of said detecting oligonucleotide, and monitoring fluorescence of said fluorescent reporter entity at least after a plurality of amplification cycles.

The invention relates to a vancomycin-resistant enterococcus multiplex fluorescence quantitative PCR (polymerase chain reaction) detection kit. The kit comprises a VanA specific primer, a VanB specific primer, a VanA fluorescent probe, a VanB fluorescent probe, a PCR buffer solution, a deoxynucleoside triphosphate mixture, a DNA (deoxyribonucleic acid) polymerase, a VanA gene standard product sequence and a VanB gene standard product sequence. The kit disclosed by the invention has the beneficial effects that due to the adoption of the PCR detection method, accurate, fast and quantitative detection can be realized.

Aspects of the invention provide novel and surprisingly effective methods for the detection of nucleic acids, comprising nucleic acid amplification using base-modified deoxynucleoside 5′-triphosphates (dNTPs). Particular aspects relate to methods for enhancing hybridization properties of oligonucleotide primers and probes in assays detecting nucleic acids, comprise amplifying target DNAs in presence of base-modified duplex-stabilizing deoxyribonucleoside 5′-triphosphates to provide for modified target DNAs, and thereby considerably improving performance of the detection assays. The disclosed methods allow for increasing of the reaction temperature in PCR-based detection systems or, alternatively, reducing the length of the oligonucleotide primers and probes. Certain aspects relates to improvement of real time PCR assays, wherein nucleic acids of interest are detected as the reaction proceeds using fluorescent agents or oligonucleotide FRET probes.

The present invention relates to a method for detecting a human leukocyte HLA-B*27 antigen gene. The method comprises: providing a mixture containing a sample requiring detection, a nucleic acid amplification system and a fluorescent detection system, adopting an amplification reaction to carry out cycle amplification of the target polynucleotide, indirectly binding a fluorescence generation group and the amplified target polynucleotide, determining the fluorescence produced by the fluorescence generation group, and determining the existence and the relative amount of the polynucleotide, wherein the nucleic acid amplification system comprises hot resistant DNA polymerase, 2'-deoxynucleoside triphosphate, and primers capable of being bound with the first chain and the second chain of the target polynucleotide. The present invention further discloses a kit matched with the detection method. With application of the method and the kit in human HLA-B*27 allele detection, the quantitative distinction of the heterozygous type and the homozygous type of the HLA-B * 27 allele can be achieved, and the advantages are provided for early warning of the HLA-B*27 allele strongly associated diseases.

The invention is concerned with a method and a composition for treating or preventing catabolism or of stimulating anabolism in a mammal undergoing metabolic stress. The method comprises administering to the mammal a composition containing methyl donors selected from the group consisting of L-serine, methionine, choline, phosphatidyl choline, betaine, dimethylglycine, sarcosine, methylated folates, S-adenosyl methionine, thymidine triphosphate, adenosine triphosphate and optionally methyl acceptors selected from the group consisting of L-glycine, ethanolamine, phosphatidyl ethanolamine, folate, ribose, wherein the total molar amount of methyl donors delivered by the method exceeds the total molar amount of methyl acceptors delivered by the method by at least 0.18 mmol per kg of body weight per day.