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Method for detecting a target nucleic acid in a sample

A technology for target nucleic acid and target detection, applied in the field of target nucleic acid detection

Inactive Publication Date: 2011-11-23
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] A drawback of fluorescent hybridization probe pairs is the high fluorescence background of the fluorescent donor dye observed when monitoring the fluorescence of the acceptor dye

Method used

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  • Method for detecting a target nucleic acid in a sample
  • Method for detecting a target nucleic acid in a sample
  • Method for detecting a target nucleic acid in a sample

Examples

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Embodiment

[0074] In these examples, the method of the present invention was used to amplify the region of the human Factor V gene containing the Leiden mutation site and cloned into a plasmid vector. In the current example, the asymmetric PCR sample master mix (100 uL) consisted of the following components: 5% glycerol; 50 mM Tricine, pH 8.3; 25 mM potassium acetate; 200 μM dATP, 200 μM dGTP, 200 μM dCTP, 400 μM dUTP; 0.7 μM upstream (excess) primer; 0.1 μM downstream (limiting) primer; 0.4 μM each probe; 0.04 U / μL uracil-N-glycosylase; 0.4 U / μL ΔZO5 DNA polymerase; and 4 mM magnesium acetate.

[0075] Master mixes are used to amplify Factor V wild-type, mutant, and mixed plasmid DNA targets. Excess primer was present at 7 times the limiting primer concentration to ensure an excess of single stranded amplicon bound to the hybridization probe. Amplification and melting were performed on a Roche LightCycler 480.

[0076] The thermal cycle profile used in this example was: 50°C for 5 min...

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Abstract

The invention relates to a method for detecting a target nucleic acid in a sample using fluorescent probe pairs which include fluorescent reporter and quencher molecules which may be used in hybridization assays and in nucleic acid amplification reactions, especially fluorescent reporter molecules and quencher molecules of polymerase chain reactions (PCR).

Description

field of invention [0001] In general terms the present invention relates to methods for detecting target nucleic acids in a sample. More specifically, the present invention relates to methods for detecting target nucleic acids in samples using pairs of fluorescent probes, including those that can be used in hybridization assays and nucleic acid amplification reactions, particularly polymerase chain reaction (PCR). Fluorescent reporter molecules and quencher molecules. Background of the invention [0002] Methods for monitoring nucleic acid amplification reactions, such as the polymerase chain reaction (PCR), using fluorescent probes are known in the art. [0003] An example of a fluorescent probe is as figure 1 Fluorescent dual-labeled probes described in . Such fluorescent dual-labeled probes generally consist of two different dyes, namely reporter molecules and quencher-labeled oligonucleotides, which can hybridize to specific target nucleic acid sequences. A reporter ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6818C12Q2565/101C12Q2527/107
Inventor N.牛顿
Owner F HOFFMANN LA ROCHE & CO AG
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