PCR Theory & Protocols
10 things that can kill Your PCR (by Peter Frame)Concept of PCR and Standardprotocol
Introduction
The idea of making unlimited DNA copies from a single copy of DNA which was later called: "Polymerase Chain Reaction" (PCR) was born in 1983 by Kary B. Mullis. Ten years later, he was awarded the Nobel Prize in Stockholm for his brilliant method.
PCR was first published in 1985 (Saiki et al., 1985). For Elongation a Klenow polymerase was used. Because Klenow polymerase is heat instable, new enzyme had to be added for every new cycle. The maximum product length was only 400 bp. In 1988 the first report using thermostable DNA polymerase from <em>Thermophilus aquaticus</em> (<em>Taq</em>-polymerase) was published (Saiki et al., 1988). PCR technique is today a powerful standard tool for molecular biology.
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. <em>Science</em>. 239:487-491.
Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, Arnheim N. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analyses for diagnosis of sickle cell anemia. <em>Science</em>. 230:1350-1354.
The principle
Double stranded DNA is denatured to single stranded DNA. A short complementary piece of DNA (the primer) could bind to the sequence of the single stranded fragment of DNA (template). The primer recognizes the template and binds (anneals) to his recognition sequence. The 3'-end of the primer is used by DNA polymerase to synthesize a new DNA strand (elongation / extension).
The three steps of one doubling cycle (Denaturation, Annealing and Elongation) depend on the temperature. So the newly-formed double stranded DNA is denatured again at 94-97ºC. By lowering the temperature the primers anneal at 35-72ºC (the exact temperature depends on the primer), and the new product is synthesized at 72ºC, which is the optimal temperature for the <em>Taq</em>-polymerase.
Multiplying identically DNA copies of template strands will take place in every repeated cycle. One cycle doubles the amount of DNA. After 25 cycles a amount of 3,2 x 107 DNA molecules is amplified. In this way the DNA sequence between the two primer sequences is amplified exponentially, resulting in high concentrations of double-stranded DNA of the same length.
