UNIT 15.1 Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
Published Online: 1 NOV 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Kramer, M. F. and Coen, D. M. 2001. Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization. Current Protocols in Molecular Biology. 56:15.1:15.1.1–15.1.14.
- Published Online: 1 NOV 2001
- Published Print: OCT 2001
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.