Oriented-polymerization fluorescent probe PCR

Abstract

The invention discloses an oriented-polymerization fluorescent probe PCR which is characterized in that conventional primer and probe are specifically combined while respective oriented polymerizationis added. A reverse base sequence at the 5' end of the opposite-side former primer is added to the front of a primer pair along the 5'-3' direction to form a '5' reverse complementary sequence' chimeric primer pair for oriented polymerization; through the 5' end complementation, the 3' tails of the amplification products serve as a template of each other and form mutual primer amplification; based on PCR hypersensitivity, the sensitivity 3-5Ct is further improved while the non-specificity is reduced, or the amplification of 5 cycles can be reduced at the same sensitivity; the 3' lengthened tail end of the fluorescent probe and the middle sequence of the probe are reversely complementary and form self-hybridization in pairs, then the quencher approaches the fluorophore group through mutualpolymerization at the tail part and the middle part so as to reduce the base line fluorescence and probe non-specificity; once a long-sequence target molecular chain is specifically hybridized with the primer and the probe chain, the pairwise polymer pair and probe with low relative competitive ability are molten and specifically combined with target molecule for amplification, and the probe is hydrolyzed by polymerase to generate fluorescence in direct proportion to the target molecule.

Description

Technical field: [0001] The invention belongs to the field of molecular biology and nucleic acid amplification PCR detection, and specifically relates to a fluorescent probe PCR technology for complementary binding of 5' ends of directional primers, sensitization and polymerization and unzipping of 3' ends of hydrolysis probes. Background technique: [0002] Nucleic acid amplification PCR technology is developed and matured together with the progress of modern molecular biology. As early as 1953, the nucleic acid DNA double helix model and semi-conservative replication opened the era of modern molecular biology and established the polymerase chain reaction. (Polymerase Chain Reaction, PCR) basis, and even Khorana directly proposed the mode of nucleic acid in vitro amplification in 1971: "After DNA denaturation, hybridize with suitable primers, use DNA polymerase to extend primers, and repeat this process continuously. tRNA genes can be cloned". In 1985, Kary Mullis of the H...

AI Technical Summary

Problems solved by technology

In view of the obvious insufficient sensitivity of fluorescent probe PCR for single-molecule detection, current sensitivity-enhancing methods are limited and require additional pretreatment steps; at the same time, they are subject to non-specific interference and inhibition of primer-primer and primer-probe aggregation, and even internal standard systems, multiplex Fluorescent probe PCR extremely complex multiplex non-specificity

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