Obtaining Maximum PCR Sensitivity and Specificity
Cameron N. Gundry and Matthew D. Poulson
from: PCR Troubleshooting and Optimization: The Essential Guide (Edited by: Suzanne Kennedy and Nick Oswald). Caister Academic Press, U.K. (2011)
PCR is a highly sensitive and specific technique used in molecular biology laboratories everywhere. It is able to provide near 100% sensitivity and specificity with appropriately designed assays in controlled situations. However, results do not always match this potential. The most common problems in PCR arise from overlooking basic principles in assay design and optimization. Maximum PCR performance depends on key factors which include: 1) choosing an appropriate detection system, 2) using available software for the best primer and probe design, 3) assessing sample quality and controlling inhibitors, 4) avoiding amplicon and environmental contamination, 5) optimizing for reagent quality and concentration, and 6) modifying the thermal cycling protocol for optimal sensitivity and specificity. This chapter will address all of these factors to aid the investigator in designing high quality PCR assays read more ...