Difficult Templates and Inhibitors of PCR
Jack M. Gallup
from: PCR Troubleshooting and Optimization: The Essential Guide (Edited by: Suzanne Kennedy and Nick Oswald). Caister Academic Press, U.K. (2011)
One of the least-acknowledged problems with PCR, RT-PCR and qPCR is reaction inhibition. Addressing or eliminating inhibition is central to allowing qPCR to be modeled by the least complex mathematics, and enables more effective troubleshooting of amplifications from difficult templates such as AT- or GC-rich sequences, repetitive sequences, and templates with prohibitive secondary structures. In the absence of inhibition, additives aimed at improving PCR, RT-PCR and qPCR performance can be assessed more directly, allowing investigators to identify and utilize better primer/probe designs, enzymes and master mixes, and formulate better reverse transcription reactions. In addition to inhibition, RNA integrity is another major concern which must be addressed both by using appropriate optical assessments and the 3':5' assay. To address inhibition, commercial kits for removing inhibitory substances have been developed in addition to the SPUD assay and the P-Q assay-development/project-management software tool. Although reagent choice alone plays a large part in determining the success or failure of reverse transcription, PCR, RT-PCR or qPCR, this chapter briefly explores some of the current strategies for detecting, avoiding and/or eliminating inhibition during reverse transcription, PCR, RT-PCR and qPCR. It also discusses strategies to amplify difficult templates and optimize reverse transcription reactions read more ...