Optimization and Troubleshooting in PCR
Open Access
- 1 April 2009
- journal article
- Published by Cold Spring Harbor Laboratory in Cold Spring Harbor Protocols
- Vol. 2009 (4), pdb.ip66
- https://doi.org/10.1101/pdb.ip66
Abstract
INTRODUCTION: The use of polymerase chain reaction (PCR) to generate large amounts of a desired product can be a double-edged sword. Failure to amplify under optimum conditions can lead to the generation of multiple undefined and unwanted products, even to the exclusion of the desired product. At the other extreme, no product may be produced. A typical response at this point is to vary one or more of the many parameters that are known to contribute to primer-template fidelity and primer extension. High on the list of optimization variables are Mg++ concentrations, buffer pH, and cycling conditions. With regard to the last, the annealing temperature is most important. The situation is further complicated by the fact that some of the variables are quite interdependent. For example, because dNTPs directly chelate a proportional number of Mg++ ions, an increase in the concentration of dNTPs decreases the concentration of free Mg++ available to influence polymerase function. This article discusses various optimization strategies, including touchdown PCR and hot-start PCR.Keywords
This publication has 16 references indexed in Scilit:
- PCR Primer DesignCold Spring Harbor Protocols, 2009
- A simple procedure for optimising the polymerase chain reaction (PCR) using modified Taguchi methodsNucleic Acids Research, 1994
- Structure and functional properties of human general transcription factor IIENature, 1991
- Enhanced evolutionary PCR using oligonucleotides with inosine at the 3′-terminusNucleic Acids Research, 1991
- Excessive cycling converts PCR products to randomlength higher molecular weight fragmentsNucleic Acids Research, 1991
- Recent Advances in the Polymerase Chain ReactionScience, 1991
- Maximizing sensitivity and specificity of PCR by preamplification heatingNucleic Acids Research, 1991
- PCR amplification of anEscherichia coligene using mixed primers containing deoxyinosine at ambiguous positions in degenerate amino acid codonsNucleic Acids Research, 1990
- Highly degenerate, inosine-containing primers specifically amplify rare cDNA using the polymerase chain reactionNucleic Acids Research, 1988
- Generation of cDNA Probes Directed by Amino Acid Sequence: Cloning of Urate OxidaseScience, 1988