Primer middle sequence interference PCR (Polymerase Chain Reaction) technology

Abstract

The invention relates to a primer middle sequence interference PCR (Polymerase Chain Reaction) technology. The improved PCR technology is characterized in that one segment of relatively non-complemented or same-sequence basic group primer molecules in the intermediate domain of primers perform the antisense interference inside and outside so as to competitively destroy the polymerization among the primers to selectively inhibit the primer dimer (PD) from being amplified. For the interference of the intermediate domain of the primers, based on the primers optimally selected by the conventional design principle, the technology that the intermediate domain (ID) of a pair of the primers are in parallel but are not complemented with each other or are in the same sequence or / and the technology that ID antisense oligonucleotides (Oligo) are added into the primers to perform the interference action or / and the Oligo antonymy is carried out in the primer molecules via the ID so as to perform the interference action are adopted, or the combined technology of the three types of the technologies is adopted. As a result, only the ID of the primers is interfered while the target specific amplification is not influenced; the combining force acting on the minority of base-group pairing hydrogen bonds at the tail end and the base-group hydrogen bonds outside the tail end due to the action of the primers is dispersed to a maximum extent, so that the PD is selectively inhibited. Therefore, the PD accumulation in the PCR system is avoided. If the mineral oil is additionally used, the sealed primers can slowly release the hot starting and the UDG pretreatment so as to prevent aerosol glue as a byproduct of the PCR system from causing the pollution. Consequently, the nucleic acid is amplified reliably and the real-time fluorescence PCR is quantified accurately.

Description

Technical field: [0001] The invention belongs to the technical field of nucleic acid amplification in the field of molecular biology and molecular testing, and specifically relates to the technical field of selectively inhibiting PCR non-specific amplification by competitively destroying polymerization between primers by interfering with the middle sequence of primers. Background technique: [0002] The idea of ​​nucleic acid amplification originated in 1971. Khorana, who discovered the genetic code, once proposed the idea of ​​nucleic acid amplification in vitro: "After DNA denaturation, hybridization with suitable primers, extension of primers with DNA polymerase, and repeated this process can clone tRNA gene" (Kleppe, 1971, J. Molec. Biol., 56:341), however, its development was limited by the lack of oligonucleotide synthesis, thermostable polymerase, and thermal cycler at that time. Until 1983, Kary Mullis of the Human Genetics Laboratory of PE-Cetus Company in the Unite...

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Problems solved by technology

Real-time fluorescent PCR amplification does not interfere with the specific amplification of the target molecule, and its background is basically a straight line within 45 cycles of the PCR reaction, without interference from non-specific amplification values

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