Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization

This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield. The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. Once assembled, the mixture is cycled many times (usually 30) through temperatures that permit denaturation, annealing, and synthesis to exponentially amplify a product of specific size and sequence. The PCR products are then displayed on an appropriate gel and examined for yield and specificity. Recommended optimization conditions are included.

[1]  K. Mullis,et al.  Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. , 1985, Science.

[2]  K. A. Eckert,et al.  High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase , 1990, Nucleic Acids Res..

[3]  L. Loeb,et al.  Taq DNA Polymerase , 2002 .

[4]  K. Mullis,et al.  Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. , 1986, Cold Spring Harbor symposia on quantitative biology.

[5]  R. Saiki,et al.  The Design and Optimization of the PCR , 1989 .

[6]  D. Birch,et al.  Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. , 1992, Nucleic acids research.

[7]  U. Gyllensten.  Direct Sequencing of In Vitro Amplified DNA , 1989 .

[8]  R. Higuchi,et al.  Simple and Rapid Preparation of Samples for PCR , 1989 .

[9]  A. Jeffreys,et al.  Amplification of human minisatellites by the polymerase chain reaction: towards DNA fingerprinting of single cells. , 1988, Nucleic acids research.

[10]  H A Erlich,et al.  Rapid prenatal diagnosis of sickle cell anemia by a new method of DNA analysis. , 1987, The New England journal of medicine.

[11]  K. Mullis,et al.  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. , 1988, Science.

[12]  Henry A. Erlich,et al.  Enzymatic amplification of ?-globin genomic sequences and restriction site analysis for diagnosis of , 1985 .

[13]  P. V. von Hippel,et al.  Betaine can eliminate the base pair composition dependence of DNA melting. , 1993, Biochemistry.

[14]  A. Chenchik,et al.  TaqStart Antibody: "hot start" PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase. , 1994, BioTechniques.

[15]  Henry A. Erlich,et al.  Analysis of enzymatically amplified β-globin and HLA-DQα DNA with allele-specific oligonucleotide probes , 1986, Nature.

[16]  H. Khorana,et al.  Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases. , 1971, Journal of molecular biology.