A PCR-LDR gene polymorphism detection and sequencing method with fluorescent labeling probe

Abstract

The invention provides a PCR-LDR gene polymorphism detection and sequencing method using a fluorescent label probe. The invention utilizes fluorescent label probes to perform PCR-associated LDR and sequencing techniques to detect gene polymorphism sites. The method of the invention comprises the following steps: primer and probe design, PCR detection, LDR detection, sequencer result detection. Due to the directness of the sequencing results, the method of the present invention solves the false positive and false negative results; the high-throughput of the samples of the sequencing platform can simultaneously detect hundreds of samples; The unique 5-color fluorescence detection system can use 4 kinds of fluorescence at the same time, so that the sequencer system can also detect more than 100 sites at the same time, speeding up the detection speed and reducing the experimental cost.

Description

technical field [0001] The invention relates to biotechnology, in particular to a PCR-LDR gene polymorphism detection and sequencing method using fluorescent labeling probes. Background technique [0002] With the development of human pharmacogenomics, more and more research results will be applied to clinical diagnosis, which requires a large-scale detection method of gene polymorphism sites. In recent years, the ligation reaction is used to detect gene polymorphic sites more and more people's attention, scholars have done a lot of research (France Barany et al., Proc.Nat'l Acad.Sci.USA, 88 : 189-93 (1991), The Ligase Chain Reaction (LCR) in a PCR World, PCR Methods and Applications, 1: 5-16 (1991)). Ligase chain reaction (ligase chain reaction, LCR) refers to the design of two pairs of probes in the reaction, so that the ligation reaction can exponentially amplify the template. In the ligase detection reaction (LDR), only a pair of probes are added to the reaction, and t...

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Problems solved by technology

[0003] At present, there have been the associated use of LDR technology and PCR technology, and the association of LDR technology, PCR technology, gene chip technology, and sequencing technology for gene polymorphism site detection (Norman P.Gerryl et al., J.Mol.Biol .(1999) 292,251-262, U.S.Pat.Pub.No.2003 / 0032016A1), the appearance of these technologies greatly facilitates the detection of gene loci, but in these technologies, a large amount of fluorescently labeled probes are required, However, these labeled probes are expensive, causing inconvenience to development and application, thus limiting their application

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