Fig. 1. The schematic concepts of duplex real-time PCR with internal standard for quantitative detection of plasma DNA. The whole process includes 4 steps: (A) A 41-bp artificial double-stranded DNA sequence corresponding to the human β–actin gene is cloned and inserted to pMD18-T vector, which is then linearized by restriction enzyme digestion. (B) The recombinant plasmid DNA is added into the cell-free plasma sample with known concentration and extracted together with endogenous nucleic acids. (C) The internal standard and target gene are then amplified simultaneously in the same tube with the common reverse primer focusing on the 41-bp corresponding sequence by duplex real-time PCR, where fluorescence signals are detected separately (Applied Biosystems 7500 Sequence Detector). (D) The plasma DNA concentrations are calculated according to the internal standard by the equation.
