34 results about "Dideoxynucleotide" patented technology

This invention provides a process for sequencing nucleic acids using 3′ modified deoxynucleotide analogues or 3′ modified deoxyinosine triphosphate analogues, and 3′ modified dideoxynucleotide analogues having a detectable marker attached to a base thereof.

Method for sequencing a nucleic acid molecule in a thermocycling reaction which initially comprises a nucleic acid molecule, a first primer, a second primer, a reaction buffer, a first thermostable DNA polymerase, (optionally) a thermostable pyrophosphatase, deoxynucleotides or derivatives thereof and a dideoxynucleotide or a derivative thereof and which is characterized in that the thermocycling reaction additionally contains a second thermostable DNA polymerase which, in comparison to the said first thermostable DNA polymerase, has a reduced ability to incorporate dideoxynucleotides as well as the use of the said method.

Cycle sequencing of DNA with marker-labeled dideoxynucleotides is performed using extremely thermophilic DNA polymerases in a one-lane technique in which dideoxynucleotides labeled with a dye as a marker are used to obtain sequence ladders of more 500 bases. Chain elongation is performed at a temperature of 65 to 75° C. Surprisingly, it was found that chain elongation temperatures of substantially more than 60° C. could be used in cycle sequencing of DNA with marker labeled dye dideoxynucleotides when using extremely thermophilic DNA polymerases.

We disclose quality controls methods that allow quick and accurate verification of a test oligonucleotide deposited on a solid support. It is especially useful for the verification of oligonucleotides representing alleles of a multi-allelic locus. It employs single base extension, with labeled dideoxynucleotides, to locate and verify the identity of test oligonucleotides. This approach involves synthesizing a complement probe oligonucleotide for each oligonucleotide being tested. Probe oligonucleotides are optionally grouped. They are then hybridized to test oligonucleotides, and the hybridized pair is subject to single base extension and detection. It requires the presence of one unique base, either in the last two bases at the free hanging end of the test oligonucleotide—as opposed to the end anchored to the solid support surface, or in the last two bases at one end of the probe oligonucleotide.

The invention discloses a method for using silkworm cultured cell to express antibacterial peptide Cecropin B. The method comprises the following steps: (1) using the total RNA extracted from the fat body cells of wild silkworm chrysalis as template, adopting RT-PCR amplification to obtain wild silkworm antibacterial peptide Cecropin B gene; using 1% agarose gel electrophoresis to perform PCR product analysis to the antibacterial peptide Cecropin B gene, using a PCR purification kit of Qiagene for purification, then cloning the purified PCR product to TA vector pCR2.1 to obtain pCR2.1-Cecropin B, utilizing the dideoxynucleotide chain termination to confirm the correctness of cloned gene order; using restriction endonuclease BamHI and HindIII to digest pCR2.1-Cecropin B and obtain Cecropin B genetic fragments, then cloning rhabdovirus transfer vector pBlueBacHisa in the genetic fragments to obtain reconstituted transfer vector; performing cotransfection of the reconstituted transfer vector and silkworm wild-type nuclear polyhedrosis virus BmNPV DNA in silkworm cultured cell, performing homologous recombination to generate recombinant virus; and (3) inoculating the recombinant virus containing wild silkworm antibacterial peptide Cecropin B gene in the silkworm cultured cell, infecting at 27 DEG C for three days to express the infection, and centrifuging to collect silkworm cultured cell.

The invention discloses a method for polymorphism detection of a gene based on single base extension reaction. The method is characterized by comprising the following steps: (1) designing and selecting a specific primer according to a gene sequence to be detected, adding the specific primer in a mixed liquid containing a sample to be detected, a dideoxynucleotide (ddNTP) pair or deoxynucleotide triphophate (dNTP) pair for single base extension reaction so that a base in ddNTP or dNTP is added at the terminal end of the specific primer, wherein the specific primer is positioned at the upstream of gene SPN (single nucleotide polymorphism) site to be detected, the 3'-terminal base of the primer is close to the SNP site to be detected, and a continuous nucleotide sequence is composed of 15-45 continuous nucleotides containing the gene sequence to be detected; and (2) detecting the amount of surplus ribonucleotides in an extended product, judging the type of the polymorphism of the target gene according to the amount of the ddNTP or dNTP in the extended product. The method has low cost and is simple to operate, and can rapidly detect the polymorphism of the gene in no need of complicated optimization steps.

The present invention relates to mutant DNA polymerases which incorporate dideoxynucleotides with about the same efficiency as deoxynucleotides. The present invention also related to mutant DNA polymerases which also have substantially reduced 5′-to-3′ exonuclease activity or 3′-to-5′ exonuclease activity. The invention also relates to DNA molecules coding for the mutant DNA polymerases, and hosts containing the DNA molecules.

The invention relates to a DNA sequencing method based on a primer array chip, and is characterized in that the method comprises the following steps: fixing a group of primers with known sequences on a chip, respectively putting four same chips into four reaction systems which contain different dideoxynucleotides with fluorescent tracers, performing annealing hybridization with DNA to be detected to allow the primers to extend 1 nucleotide, detecting the fluorescence signals of the chips to obtain a group of short sequences composed of primer end sequences and extended nucleotides, splicing the short sequences overlapped with each other to obtain a complete sequence of the DNA to be detected.

A method is described for the direct, exponential amplification and sequencing ("DEXAS") of a DNA molecule from a complex mixture of nucleic acids, wherein truncated DNA molecules as well as DNA molecules of full length are synthesized simultaneously and exponentially between two positions on the said DNA molecule, which initially contains a DNA molecule in a thermocycling reaction, a first primer, a second primer, a reaction buffer, a thermostable DNA polymerase, a thermostable pyrophosphatase (optionally), deoxynucleotides or derivatives thereof and a dideoxynucleotide or derivatives thereof. In a preferred embodiment of the method of the invention, direct sequencing of RNA can be performed using one polymerase having a Tabor-Richardson mutation, or a functional derivative thereof, and reverse transcriptase activity. In a more preferred embodiment of the method of the invention, direct sequencing of RNA can be performed in one step, in one vessel.

A method for detecting amplified nucleic acid fragments is provided. The method comprises the steps of carrying out amplification reaction to amplify a predetermined region of a nucleic acid in a reaction solution comprising the nucleic acid as a template, a pair of primers, deoxynucleotides, and dideoxynucleotides; hybridizing the amplified products of the reaction solution to a probe; and then detecting the hybridization between the amplified products and the probe.

The invention provides a preparation method and application of an oligonucleotide with a closed dideoxyribonucleoside. The method specifically comprises the steps that a terminal transferase (TdT) isfirstly used for catalyzing deoxyribonucleotides (dNTPs) or dideoxynucleotides (ddNTPs) in combination with the characteristics of a 3'hydroxyl terminal of a DNA molecule, so that a primer is mixed with any one of four dideoxynucleotides (ddATP, ddTTP, ddCTP or ddGTP), the TdT can add the dideoxynucleotides to the 3'hydroxyl terminal of the primer, and the obtained primer modified by the ddNTP cannot be catalytically extended by a DNA polymerase. The prepared oligonucleotide (primer) with the closed dideoxyribonucleoside can be used in combination with amplification methods such as allele PCR,proof-reading PCR for detecting mutation.

A method for sequencing a nucleic acid strand, comprising the steps of: providing a solution containing truncated strands having lengths different from one another terminating with a respective dideoxynucleotide from among ddATP, ddTTP, ddGTP, and ddCTP; functionalizing first masses by a donor molecule and second masses by an acceptor molecule such as to generate a light emission when they come into mutual contact; coupling a first mass to a first end of each truncated strand; coupling the second masses to a respective terminal dideoxynucleotide of each strand; applying an AC electrical field having variable frequencies that are such as to generate, on each second mass, a net movement directed towards the first mass; acquiring a plurality of light radiations for each frequency value; and associating each light radiation acquired to a respective dideoxynucleotide and, thus, to a respective nucleotide base.

Disclosed is a method whereby a repetitive nucleic acid sequence, such as a short tandem repeat (STR), may be characterized as to its length. Pyrosequencing is used to sequence an STR repetitive region to measure the length of STRs in a rapid manner. A combinatorial approach is disclosed for the addition of multiple nucleotides (e.g., two mononucleotides) at a time by the polymerase, which reduces the sample analysis time by half. In addition, modified nucleic acids, such as peptide nucleic acids, are used as blocking probe to stop polymerization on the flanking region which makes it possible to use pyrosequencing for DNA length measurement both in the case of homozygous or heterozygous samples for varying repeat patterns of different markers. Further, dideoxynucleotides are added to stop polymerization in the flanking region of the STR.