Increased specificity of rhPCR with complex DNA samples. A PCR assay specific for the human HRAS gene was used to compare the specificity of "rDDDDx" blocked-cleavable primers (rhPCR) with unmodified control primers to amplify the desired sequences in human cDNA versus mismatched sequences in rat cDNA. A. Amplification plots are shown for SYBR® Green qPCR assays run with unmodified control primers (left panel) or blocked-cleavable primers (right panel). Reactions with human cDNA (HeLa) are shown in black and reactions with rat cDNA (spinal cord) are shown in blue. The concentration of RNase H2 was 1.3 mU per 10 μL and the anneal/extension time was 90 seconds. B. The human HRAS-specific amplification primers are shown aligned with the homologous sequence in the rat Hras gene. Unmodified control primers are shown on the left and blocked-cleavable primers are shown on the right. DNA bases are black uppercase and RNA bases are red lowercase. "x" is a propanediol C3 spacer.
Dobosy et al. BMC Biotechnology 2011 11:80 doi:10.1186/1472-6750-11-80