69 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.

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 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.