Reverse complementary fluorescent PCR at 5' ends of primers

Abstract

The invention discloses reverse complementary fluorescent PCR at 5' ends of primers and belongs to the technical field of molecular biology-nucleic acid amplification. The reverse complementary fluorescent PCR is characterized in that a segment of sequence reverse complementary fluorescent PCR is added at the 5' ends of a pair of primers for amplifying, so that tail ends of amplified products arecomplementary; 3' ends of target products also can be templates and primers with each other, so that amplification efficiency is increased; the sensitivity is improved by 50 times on the basis of enlarging a conventional PCR index by 109-12, or 5 cycles of amplification can be reduced under the condition of same sensitivity; on the other side, complement of the 5' ends of the primers cannot extendrather than inhibit polymerization nonspecificity between 3' ends of a primary primer pair or 5 cycles of amplification are reduced, so that an anonspecific amplification region can be kept off. Themiddle same sequence primer taking the reciprocal first to second base adenine A at the least significant end of 3' as a tail and UDG enzymolyzed dU are combined to replace an amplification product ofdT; in addition, by closing mineral oil, no primer dimer PD is produced in a PCR reaction cycle, and cross contamination of product aerosol adhesive is avoided.

Description

Technical field: [0001] The invention belongs to the technical field of fluorescent PCR for molecular biology and nucleic acid detection, and specifically increases the complementary sequence of the 5' end of the primer to exceed the exponential amplification to improve the sensitivity and limit the non-specificity of the single-tube one-step real-time fluorescent PCR. Background technique: [0002] Polymerase chain reaction PCR is based on the DNA double helix model and semi-conservative replication rules established by Watson in 1953. It is a nucleic acid amplification technology that simulates natural gene replication in vitro (in test tube). As early as 1971, Khorana had published a nucleic acid in vitro Amplification (J.Molec.Biol., 56:341) related papers; but it was not until 1985 that Mullis of Cetus Company of the United States thought of the great power of the essence of PCR-exponential amplification or geometric progression amplification, and invented a pair of spec...

AI Technical Summary

Problems solved by technology

[0010] Therefore, the sensitivity of conventional PCR and real-time fluorescent PCR to single-molecule detection is still slightly insufficient, and ultra-sensitive amplification is needed to further improve the system sensitivity; however, PCR is limited to non-specific coverage of low-concentration sample detection, and ultra-sensitive PCR is even effective for single molecules. Detection depends on two aspects of PCR sensitization and non-specific removal

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