DTT quenches the passive reference signal in real-time PCR

Abstract

Forensic DNA analysis is partly limited by PCR-inhibitory compounds present in the DNA extracts. Generally, these inhibitors disturb amplification, i.e., the production of amplicons. We have found that dithiothreitol (DTT) from the DNA extraction process can cause another type of real-time PCR disturbance, i.e., inhibition of signal detection through fluorescence quenching. DNA extracts containing DTT substantially quenched the passive reference signal in the Quantifiler HP DNA Quantification kit. This quenching resulted in overestimation of DNA concentrations, as target DNA signals are normalized to the passive reference signal.