A method for reducing the dimer of a pair of partial homosequence primers

Abstract

The invention discloses 'a method for reducing dimers of a pair of primers with part of sequence being identical'. According to the discovery of the inventor, in a primer pair without continuous complementary bases, the resultant force of multiple spaced hydrogen bonds formed by parallel pairing in a direction from 5' to 3' promotes some complementary bases at the 3' terminals of the primer pair to form hydrogen bonds that bond and extend the terminals to form a dimer; and a single primer does not form a dimer by itself. Similar single primer of a polymerase chain reaction (PCR) primer pair is designed according to a proportion as high as possible. The method is characterized in that: two fragments of identical sequences containing 4 to 10 bases each are present in the middle of the PCR primer pair along the direction from 5' to 3', wherein the 1 to 5 bases in the 3' terminal of each primer do not contain spaced complementary bases; each 5' terminal region may have not more than three spaced complementary bases; and preferably, the sequences at the 5' terminal are identical completely. The method can greatly reduce the formation of the dimers of the primers and optimize the quality of PCR, particularly real-time PCR of a fluorescent dye.

Description

Technical field: [0001] The invention belongs to the field of molecular biology technology and nucleic acid amplification PCR technology, and in particular relates to a primer design method for reducing the formation of primer dimers. Background technique: [0002] Nucleic acid research has gone through more than half a century of exploration since the establishment of the genetic material basis as ribonucleic acid DNA. In 1953, Watson and Crick established the DNA double helix structure model and clarified its central dogma, thus opening the molecular biology of nucleic acid research. era. Since all kinds of nucleic acid DNA are linear structures composed of four base arrangements that only encode genetic information, and their physical and chemical properties are basically the same, it is difficult to separate and purify trace target genes or specifically detect trace target genes through general physical and chemical techniques. In the 1970s and 1980s, the Cold Spring Ha...

AI Technical Summary

Problems solved by technology

Since all kinds of nucleic acid DNA are composed of four base arrangements and only encode different linear structures of genetic information, their physical and chemical properties are basically the same, so it is difficult to separate and purify trace target genes or specifically detect trace target genes through general physical and chemical techniques

In the 1970s and 1980s, the Cold Spring Harbor Laboratory in the United States developed a complete system of molecular cloning technology based on bacterial monoclonal screening of gene libraries and transformation genes. However, obtaining the target gene is still time-consuming and cumbersome.

However, these bioinformatics software only solve the above general optimization conditions. Using these principles, the primer pairs designed by the application software without continuous complementary sequences are optimized in terms of specificity, length, GC content, annealing temperature Tm value and secondary structure. In practical work, the synthetic primer pairs always have primer-dimer problem more or less, and there is no regularity, the repeatability of the test results is poor, and the fundamental problem that the optimized primer pair still forms dimers has not yet been publicly reported.

The formation of primer dimers not only produces a high noise background, but also seriously interferes with the quantitative accuracy or weak positive judgment of real-time fluorescent PCR low copy (copy) samples using the fluorescent dye SYBRGreen I; it also competitively interferes with the specific amplification of the template. Increase efficiency, making most real-time fluorescent quantitative PCR techniques quantitatively inaccurate

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