PCR method of universal primer and its reaction liquid and application in multiple PCR

Abstract

A PCR method for universal primer, its reaction liquid, and its application in multiple PCR are disclosed. Said PCR method features that a pair of leader primers complementary with template in region 3' and a pair of universal primers the same as said leader primer in region 5' are amplified in a single PCR. Its advantages are high reaction specificity, stable amplifying efficiency and wide concentration range of primer.

Description

technical field [0001] The invention relates to molecular biology technology, in particular to a polymerase chain reaction method, its reaction liquid and its application in multiple PCR. technical background [0002] PCR is a simple, specific, sensitive, and rapid gene amplification method, and its process usually includes 20-40 cycles consisting of three steps of denaturation, annealing, and extension. The key to the PCR method is to design a pair of primers with high stringency that are complementary to the two strands of the DNA of the target gene according to the sequence of the target gene. The double-stranded DNA is unwound into two single strands at the denaturing temperature. When the temperature drops to the annealing temperature, the forward and reverse primers are respectively combined with the two strands of the template DNA, and then at the extension temperature, the DNA polymerase starts at the 3' end where the primer and the template bind. One cycle of ampli...

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Problems solved by technology

[0004] Existing multiplex PCR uses multiple pairs of primers that match the base sequence of the target template to simultaneously add to a PCR reaction tube to achieve multiple amplification. During the entire PCR amplification process, primers that are complementary to the original template are always used. In fact, , each pair of primers is paired with the template and then extended, each PCR is not related to each other, because the structure of the primer itself, the efficiency of binding to the template, and the change of the primer concentration in the whole reaction process are quite different among each pair of primer pairs, the amplification of each PCR Efficiency cannot be consistent, and even some defects that "weak PCR" are covered by "strong PCR" will be produced, and insufficient products will not be produced.