An Analysis of Efficiency and Melt Curve Effects on Quantitative Polymerase Chain Reaction (qPCR) Inhibition

• Main Author: Thompson, Robyn E
• Format: Others
• Published: FIU Digital Commons 2010
• Subjects: qPCR; Inhibition; DNA; Binding; Melt; Amplification; Efficiency; Forensic
• Online Access: http://digitalcommons.fiu.edu/etd/329; http://digitalcommons.fiu.edu/cgi/viewcontent.cgi?article=1408&context=etd

Real-time or quantitative PCR (qPCR) is an innovative method used to determine the amount of amplifiable DNA in a biological sample. Typically, a fluorescent dye is introduced during thermal cycling, causing a change in fluorescent output as the double-stranded DNA (dsDNA) product accumulates. Both TaqMan® and Plexor HY System methods detect PCR inhibition through the monitoring of internal control sequences. Alternatively, SYBR®Green and Plexor detect inhibition through melt curve effects. Previous work using SYBR®Green intercalation has demonstrated that inhibitors can affect melt curves differently depending on their structure and mode of action. Inhibitors that bind DNA can cause melt curve shifts while those primarily affecting Taq polymerase do not. Unlike SYBR®Green, Plexor dyes are fluorescently linked to a modified base, 5'-methylisocytosine (iso-dC), adjacent to the 5' end of the dsDNA. This produces minimal interference in dsDNA structure making it an ideal procedure for measuring these effects. In this study, inhibition of qPCR was evaluated by observing the effects of various inhibitor concentrations and amplicon lengths on DNA amplification.