Taqman real-time fluorescence PCR (polymerase chain reaction) capable of reducing polymerization with primers

Abstract

The invention provides a Taqman real-time fluorescence PCR (polymerase chain reaction) capable of reducing polymerization with primers. The Taqman real-time fluorescence PCR is improved Taqman and real-time fluorescence PCR which is in a hybrid stem structure combining a panhandle shape at the tail end of a molecular beacon and improves self combination. The Taqman real-time fluorescence PCR is characterized in that: fluorescein is labeled conventionally at 5'-terminal of a template probe sequence, complementary sequences of 5'-terminals of 5 to 6 probes are added to 3'-terminal of the template probe sequence, and a quencher is labeled at the tail terminal so as to form a stem structure similar to the tail terminal of the molecular beacon by hybridizing with the sequence at 5'-terminal of the probe. Two terminals of the probe are self-hybridized and combined, so that the quenching effect is increased, and the background is reduced; the probe is difficultly polymerized with a PCR primer due to self combination, so that primer-probe nonspecific polymerization amplification is eliminated, the detection sensitivity of the Taqman real-time fluorescence PCR is improved, and especially the relevance ratio of a weak-positive specimen is increased.

Description

Technical field: [0001] The invention belongs to the technical field of molecular biology and nucleic acid amplification PCR, and specifically relates to a real-time fluorescent PCR technology of a hydrolysis probe that reduces or even does not polymerize with primers. Background technique: [0002] Nucleic acid amplification PCR technology is developed and matured together with the progress of modern molecular biology. As early as 1971, Korana proposed the idea of ​​nucleic acid amplification in vitro: "After DNA denaturation, hybridize with suitable primers, use DNA The polymerase extends the primer, and the process is repeated to clone the tRNA gene." In 1983, Kary Mullis of the Human Genetics Laboratory of PE-Cetus Company in the United States had the inspiration of nucleic acid amplification when studying nucleic acid sequencing methods: to simulate the natural DNA in vivo replication process in test tubes. Provide a suitable condition---template DNA, oligonucleotide p...

AI Technical Summary

Problems solved by technology

What's more serious is that probes such as TaqMan and excess primers will also hybridize, complement and extend. When no template is added, the Ct value of SYBR Green I real-time PCR with only a pair of primers and TaqMan probes is advanced to about 25 cycles. It shows that the primer plus probe PCR will form some "dimers and trimers with probe sequences", and then the non-specifically amplified "dimers and trimers" compete for binding to a large number of TaqMan probes, making it difficult for weak positive templates to bind probes that may miss

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