CH391L/S12/PCR and advanced PCR techniques

What's PCR?

PCR stands for polymerase chain reaction, which is powerful technique used for in vitro exponential amplification of a specific DNA region (target region)that lies between two regions (primers) of known DNA sequence, resulting in a large quantity of DNA (~ micrograms of DNA).

Short history of PCR

The invention of PCR has long been considered as an "Eureka" event occurred on Kary Mullis' travel to his cabin[Eureka moment]. However, it would not happen if the following milestones in molecular biology had not taken place:

• Complementary double-stranded DNA model proposed by Watson and Crick [1].
• Studies on DNA replication and polymerase by Arthur Kornberg in 1950s [2].
• Klenow fragment from E. coli [3].

Interestingly, Khorana and his student had two very close chances to make this invention under their names: the first one is their project "repair synthesis" [4]. They applied primer, template and polymerase in the in vitro system. They repeatedly adding the polymerase every cycle. However, there was only one primer, and therefore, no accumulation for the DNA product. The second chance are the following words from Khorana's student Kleppe, which is famous for its involvment in the PCR patent lawsuit [5]., failed to give any substantial bench proof:

References

1. WATSON JD and CRICK FH. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature. 1953 Apr 25;171(4356):737-8. DOI:10.1038/171737a0 |
2. LEHMAN IR, BESSMAN MJ, SIMMS ES, and KORNBERG A. Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J Biol Chem. 1958 Jul;233(1):163-70.
3. Klenow H and Henningsen I. Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. Proc Natl Acad Sci U S A. 1970 Jan;65(1):168-75. DOI:10.1073/pnas.65.1.168 |
4. Kleppe K, Ohtsuka E, Kleppe R, Molineux I, and Khorana HG. Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases. J Mol Biol. 1971 Mar 14;56(2):341-61. DOI:10.1016/0022-2836(71)90469-4 |

5. ISBN=0-817-63750-8