These three stages comprise one cycle. The number of cycles of PCR required for amplification depends upon the
number of copies of DNA template present at the beginning of the reaction and the efficiency of the primer extension
and amplification. As each cycle is completed there is an exponential accumulation of a specific fragment whose
ends are determined by the 5'ends of the primers (See Figure 2).
PCR can proceed until one of the essential components becomes limiting.
The essential components of PCR are:
(1) a thermostable DNA polymerase
(2) a pair of synthetic primers
(3) deoxynucleotide triphosphates (dNTPs)
(4) reaction buffer to maintain pH (typically containing Tris and KCl)
(5) divalent cations (most commonly Mg2+)
(6) template DNA.
Figure 2. Exponential Accumulation of Fragments.
Varying any one of these components, or the cycling parameters including cycle number and annealing temperature,
affects the specificity and/or yield of the PCR reaction.
PCR success heavily depends upon the individual template-primer combinations. Before beginning PCR, preliminary
experiments to optimize reaction conditions are essential. Conditions for optimization may include variation of the
cycling parameters and/or concentrations of essential reaction components. Additives (i.e. DMSO or betaine) are
also useful when dealing with difficult templates such as GC rich sequences. For more information, see our PCR
Troubleshooting Tips on the web.
Pierce Nucleic Acid Technologies offers a variety of products featured within this application note. Table 1 summarizes these products.
References
R. A. Eeles and Stamps, A. C. Polymerase Chain Reaction (PCR) the Technique and its Applications. CRC Press, Austin, TX. 1993.
R.G. Landes Company. Pages 4-25.
M.J. McPherson, B.D. Hames, and G.R. Taylor. (eds.) PCR 2: A Practical Approach. IRL Press, N.Y., New York. 1995. Oxford
University Press. Page 5.
A. Rolfs; Shculler, I.; Finckh, U.; and Weber-Rolfs, I. PCR: Clinical Diagnostics and Research. Springer-Verlag, Berlin Germany.
1992. Springer-Verlag Berlin Heidelberg. Pages 51-60.
J. Sambrook and Russell, D. W. Molecular Cloning: A Laboratory Manual. Vol. 2. Third edition. Cold Spring Harbor Press, Cold
Spring Harbor, New York. 2001 Cold Spring Harbor Press, Cold Spring Harbor, New York. Pages 8.1-8.126.
