Cutting out the amplicon with restriction endonuclease – a way to exclude the false results of RT PCR with TaqMan probe
Akishev A.G., Netesova N.A., Abdurashitov M.A., Degtyarev S.Kh.
Real-time PCR with TaqMan probe (RT PCR) is one of the main methods for analyzing the structure of human DNA and is widely used in medicine. In this work, PCR RT of 4 fragments of human genomic DNA (chr1: 90717178-90717300, chr9: 97464604-97464756, chr11: 65647215-65647307, chr17: 4560027-4560175) was performed with DNA preparations from human blood obtained both by the standard method of phenol deproteinization and isolated on columns. Preparations of the original DNA and DNA after a treatment with a restriction endonuclease that cuts out the amplified fragment were used in PCR. In the case of native DNA preparations purified by column, PCR of 3 from 4 DNA fragments provided an overestimated Cq value. In the case of the phenol purification, an overestimated Cq value for native DNA preparations was obtained for 2 from 4 DNA fragments. For DNA preparations obtained by the column purification, treatment of DNA with a restriction endonuclease that cuts out the amplified fragment leads to normal Cq values for all four analyzed DNA fragments. Thus, RT PCR of native DNA obtained by the column purification may result in an overestimation of Cq, which is avoided if DNA previously has been hydrolyzed by a restriction endonuclease that cuts out the amplicon.