Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR.

ABSTRACT: Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded

SUBMITTER: Sidstedt M 

PROVIDER: S-EPMC5857286 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature