Support & Resources


Culprits in whole blood affecting quantitative PCR analysis: quenching of EvaGreen® fluorescence by haemoglobin

Direct PCR analysis from whole blood, without DNA extraction, is very appealing in field studies and in routine analysis of clinical, genetic or forensics samples. It offers substantial savings in cost and time and simplifies sample processing. Amplification of DNA templates from whole blood, however, remains challenging. Obtaining reliable and quantitative results is hampered by the presence of inhibiting substances.

Previous studies largely used conventional PCR to characterize the inhibitory mechanisms of blood and blood components. A recent publication aims to address this issue in the context of more quantitative methods: real-time PCR (qPCR) and digital PCR (dPCR). Hemoglobin and IgG were found to be the main offenders. Haemoglobin affects amplification through a direct effect on the DNA polymerase activity. Additionally, the authors show that haemoglobin also quenches of fluorescence of EvaGreen® and the passive reference dye ROX, leading to failed detection of amplicons. IgG, on the other hand, binds ssDNA templates and delays amplification in early PCR cycles, potentially by hindering primer annealing or binding of DNA polymerase, thus disturbing the initiation of amplification, resulting in elevated Cq values.