Fluorescent constant-temperature amplification technique

Abstract

The invention discloses a fluorescent constant-temperature amplification technique. In water used as solvent for a fluorescent constant-temperature amplification reagent used in the technique, Tris-buffer solution, magnesium acetate, dithiothreitol, betaine, Tween 20, DMSO, mycose, PEG, BSA, dNTPs, and proper amounts of fluorescent dye, polymerase, single strand DNA-binding protein and recombinase are added. A constant-temperature amplification system of the invention is dependent of high-energy molecules such as ATP and creatine phosphate; compared with existing constant-temperature amplification systems, the constant-temperature amplification system is simpler in composition, lower in cost and more convenient to use. The constant-temperature amplification system is capable of amplifying DNA and RNA sequences, is applicable to amplification of complex templates stably and quickly, capable of finishing an amplification reaction within 30min, has high amplification sensitivity and accuracy without rare occurrence of mispairing, and has good amplification effect. The constant-temperature amplification system is applicable to real-time quantification and has a wider range of application.

Description

technical field [0001] The invention relates to a fluorescent nucleic acid constant temperature amplification technology, in particular to a fluorescent constant temperature amplification technology independent of high-energy energy molecules. Background technique [0002] In 1983, Mullis et al. invented the polymerase chain reaction (PCR) technology. Over the past 30 years, PCR technology has been greatly developed. Due to its advantages in sensitivity and specificity, PCR technology has been rapidly applied to scientific research and clinical research. every aspect. This PCR technology, which is similar to the natural replication process of DNA, relies on oligonucleotide primers complementary to both ends of the target sequence. It mainly includes three basic reaction steps: (1) Denaturation of template DNA: After heating for a certain period of time, the double-stranded DNA of the template or the double-stranded DNA formed by PCR amplification is dissociated and becomes ...

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Problems solved by technology

[0003] There are also several limitations in the technical application of PCR: first, PCR technology requires expensive laboratory instruments, and at the same time, the instruments must have sophisticated temperature control procedures and heating templates, so as to realize the denaturation of DNA template strands at very high temperatures. The primer probe anneals to extend the template at a low temperature, so that the amplification of the template amount is achieved after repeated temperature changes for dozens of cycles

Since each cycle time is short, the instrument must be able to quickly and accurately raise and lower the temperature, which requires a silver or gold heating module, which increases the cost of instrument development

Second, the useful polymerase in the PCR amplification system must have the ability to withstand high temperature, otherwise new enzymes must be added in each cycle to achieve the amplification of the next cycle, which is very easy to cause pollution and increase cost

The reaction system of the existing constant temperature amplification technology is relatively complicated. At the same time, most of the constant temperature amplification systems either rely on ATP, creatine kinase, creatine phosphate, or other high-energy energy molecules, and the required reagents are various. , the amplification speed is limited by the energy supply, which leads to relatively high cost of constant temperature amplification technology, which limits its application

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