RT-PCR method integrated with restriction endonuclease removal of DNA pollution

Abstract

The invention provides a RT-PCR method integrated with restriction endonuclease removal of DNA pollution. According to the method, sequence of a restriction endonuclease recognition site on target RNA is selected as a RT-PCR amplification region. After reverse transcription, enzyme digestion reaction and PCR reaction are integrated in a system and are successively and continuously carried out. cDNA sequence and target RNA form RNA / DNA heteroduplex which will not be cut, trace amounts of genome DNA or cross-contaminated PCR amplification products are all double-stranded DNA which will be cut, and restriction endonuclease is inactivated during the PCR pre-denaturation process. Thus, cross contamination and interference of trace amounts of genome DNA or amplification products are avoided, and PCR amplification efficiency of cDNA will not be influenced. By the adoption of the method, DNase enzyme treatment is not required during the RNA sample preparation process, and cross contamination of PCR products is also greatly reduced.

Description

(1) Technical field [0001] The invention relates to an RT-PCR method for removing DNA contamination by integrating restriction endocutting. (2) Background technology [0002] The biggest feature of PCR reaction is that it has great amplification ability and high sensitivity, but the troublesome problem is that it is easy to be polluted, and an extremely small amount of pollution can cause false positives. Cross-contamination of PCR amplification products is the main and most common contamination problem in PCR reactions. The most likely form of PCR product contamination is aerosol contamination. Because the PCR product has a large number of copies (usually 10 13 copy / ml), which is far higher than the limit of PCR detection of several copies, so a very small amount of PCR product contamination can cause false positives. Contamination mainly occurs during sample preparation and system preparation. Since RT-PCR is generally carried out in two tubes for RT reaction and PCR r...

AI Technical Summary

Problems solved by technology

The disadvantages are: (1) The application is limited. It should be noted that the DNA incorporated with dUTP should not affect any operation of the product. When cloning the PCR product, it should be transformed into UNG- (UNG-deficient) E. coli recipient bacteria, otherwise The transformation product will be digested by the recipient strain UNG

(2) The use of UNG, and the required dUTP or primer incorporation of dU, greatly increases the cost

Implementation of this method requires the use of ultracentrifugation, which limits generalizability

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