Optimization and troubleshooting in PCR.

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.

[1]  E. Beutler,et al.  Interference of heparin with the polymerase chain reaction. , 1990, BioTechniques.

[2]  J. Lüthy,et al.  Improving PCR efficiency , 1990, Nature.

[3]  J. Mannhalter,et al.  Nested primer PCR detection limits of HIV-1 in the background of increasing numbers of lysed cells. , 1994, BioTechniques.

[4]  S. Roberds,et al.  Highly degenerate, inosine-containing primers specifically amplify rare cDNA using the polymerase chain reaction. , 1988, Nucleic acids research.

[5]  D. Bell,et al.  Excessive cycling converts PCR products to random-length higher molecular weight fragments. , 1991, Nucleic acids research.

[6]  K. Mullis,et al.  Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. , 1987, Methods in enzymology.

[7]  B. Cobb,et al.  A simple procedure for optimising the polymerase chain reaction (PCR) using modified Taguchi methods. , 1994, Nucleic acids research.

[8]  M. Batzer,et al.  Enhanced evolutionary PCR using oligonucleotides with inosine at the 3'-terminus. , 1991, Nucleic acids research.

[9]  C. Dieffenbach,et al.  A computer program for selection of oligonucleotide primers for polymerase chain reactions. , 1990, Nucleic acids research.

[10]  J F Medrano,et al.  Organic solvents as facilitators of polymerase chain reaction. , 1991, BioTechniques.

[11]  J. Mattick,et al.  'Touchdown' PCR to circumvent spurious priming during gene amplification. , 1991, Nucleic acids research.

[12]  G. Ruaño,et al.  Heat-soaked PCR: an efficient method for DNA amplification with applications to forensic analysis. , 1992, BioTechniques.

[13]  R. Horton,et al.  Gel-loading dyes compatible with PCR. , 1992, BioTechniques.

[14]  C. Ou,et al.  Use of UV irradiation to reduce false positivity in polymerase chain reaction. , 1991, BioTechniques.

[15]  R. Tjian,et al.  Structure and functional properties of human general transcription factor IIE , 1991, Nature.

[16]  F. Defilippes.  Decontaminating the polymerase chain reaction. , 1991, BioTechniques.

[17]  Lee Ab,et al.  Improved direct PCR screen for bacterial colonies: wooden toothpicks inhibit PCR amplification. , 1995 .

[18]  S. T. Isaacs,et al.  Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction , 1991, Nucleic Acids Res..

[19]  M K Viljanen,et al.  Primers are decisive for sensitivity of PCR. , 1994, BioTechniques.

[20]  R. T. D'Aquila,et al.  Maximizing sensitivity and specificity of PCR by pre-amplification heating , 1991, Nucleic Acids Res..

[21]  U. Linz.  Thermocycler temperature variation invalidates PCR results. , 1990, BioTechniques.

[22]  D. Labuda,et al.  Use of γ irradiation to eliminate DNA contamination for PCR , 1990 .

[23]  B. Swaminathan,et al.  Effect of ionic and nonionic detergents on the Taq polymerase. , 1990, BioTechniques.

[24]  G. Sarkar,et al.  More light on PCR contamination , 1990, Nature.

[25]  S. Kwok,et al.  Avoiding false positives with PCR , 1989, Nature.

[26]  K. Roux.  Using mismatched primer-template pairs in touchdown PCR. , 1994, BioTechniques.

[27]  R. Gibbs.  DNA amplification by the polymerase chain reaction. , 1990, Analytical chemistry.

[28]  K. Roux,et al.  High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. , 1996, BioTechniques.

[29]  K. Mullis.  The polymerase chain reaction in an anemic mode: how to avoid cold oligodeoxyribonuclear fusion. , 1991, PCR methods and applications.

[30]  J. Hartley,et al.  Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. , 1990, Gene.

[31]  J. Sninsky,et al.  Recent advances in the polymerase chain reaction , 1991, Science.

[32]  H. Seifert,et al.  Paraffin beads can replace mineral oil as an evaporation barrier in PCR. , 1993, BioTechniques.

[33]  K. Jeang,et al.  Long-range jumping of incompletely extended polymerase chain fragments generates unexpected products. , 1994, BioTechniques.

[34]  G. Caetano-Anollés,et al.  Automated "hot start" PCR using mineral oil and paraffin wax. , 1993, BioTechniques.

[35]  R. Gibbs,et al.  Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase. , 1988, Science.

[36]  W. Rychlik,et al.  A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. , 1989, Nucleic acids research.

[37]  R. Patil,et al.  PCR amplification of an Escherichia coli gene using mixed primers containing deoxyinosine at ambiguous positions in degenerate amino acid codons. , 1990, Nucleic acids research.

[38]  S. T. Isaacs,et al.  Post-PCR sterilization: development and application to an HIV-1 diagnostic assay , 1991, Nucleic Acids Res..

[39]  M. Ehrlich,et al.  Detection and quantitation of low numbers of chromosomes containing bcl-2 oncogene translocations using semi-nested PCR. , 1994, BioTechniques.

[40]  A M Prince,et al.  PCR: how to kill unwanted DNA. , 1992, BioTechniques.

[41]  K. Wilson,et al.  UV absorption complicates PCR decontamination. , 1992, BioTechniques.