One-stop PCR (Polymerase Chain Reaction) method

Abstract

The invention relates to a novel PCR (Polymerase Chain Reaction) reaction method belonging to the field of biochemistry and molecular biology. The novel PCR reaction method sequentially comprises the following steps of: (1) preparing a compatible dye mixture, wherein the compatible dye mixture comprises ficoll 400, cresol red, lemon yellow and Gelred; (2) preparing a PCR principal mixture, wherein the PCR principal mixture comprises the compatible dye mixture, dNTP (Deoxy-ribonucleoside Triphosphate), a Taq DNA polymerase and a reaction buffer solution; (3) configuring a PCR reaction system; (4) setting and operating PCR reaction conditions; and (5) detecting PCR products. The novel PCR reaction method realizes the direct point sample electrophoresis and the immediate dyeing after PCR reaction; besides, the novel PCR reaction method uses a nontoxic nucleic acid dye and has low cost and high efficiency without toxicity; the obtained PCR products can be used for directly carrying out multiple downstream operations, such as ethanol precipitation, silica gel membrane product purification, and the like; and in addition, the novel PCR reaction method is suitable for the research of gene expression detection related to PCR, gene clone, and other aspects.

Description

technical field [0001] The invention relates to a new method for nucleic acid amplification and detection, which belongs to the technical field of molecular biology. Background technique [0002] Polymerase chain reaction (polymerase chain reaction, PCR) has been invented by Dr. Karry Bank Mullis, an American scientist and Nobel Prize winner in chemistry since 1990 (Mullis, K.B.1990. The unusual origin of the polymerase chain reaction. Sci Am 262, 56- 61.), because it can achieve exponential replication of target gene fragments in a short period of time, it has now become the most important means of in vitro nucleic acid amplification, and is widely used in various fields of biological research. [0003] On the basis of the "PCR" concept proposed by Dr. Mullis, scientists have explored and developed dozens of PCR derivative technologies, such as recombinant PCR, asymmetric PCR, in situ PCR, etc., which greatly facilitate biological researchers Carry out various research act...

AI Technical Summary

Problems solved by technology

As an important conventional molecular biology technology, PCR often encounters the following problems in practical application: (1) the amplification efficiency of PCR reaction is not high enough; (2) the sensitivity of PCR amplification is limited; (3) in the When conducting experiments such as large-scale library screening, the step of adding sample buffer and mixing samples will consume a lot of time and energy; (4) dyes used for nucleic acid staining such as EB, SYBR Green, etc. are toxic, not only directly endangering the experiment The health of personnel also causes different degrees of environmental pollution (Huang, Q., and Fu, W.L. Comparative analysis of the DNAstaining efficiencies of different fluorescent dyes in preparative agarose gelelectrophoresis. Clin Chem Lab Med 2005, 43, 841-842. )

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