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 very close chance to make this invention under their names: 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. Even Khorana's student Kleppe wrote following words on the paper of this project, which is famous for its involvment in the PCR patent lawsuit [5], but failed to give any substantial bench proof thereafter:

"... one would hope to obtain two structures, each containing the full length of the template strand appropriately complexed

with the primer. DNA polymerase will be added to complete the process of repair replication. Two molecules of the original

duplex should result. The whole cycle could be repeated, there being added every time a fresh dose of the enzyme." [4]

Needless to say, Kary Mullis is the inventor of today's PCR technique. And this has to go back to 1979 when he was hired by Cetus Corporation(though PCR patent later sold to Roche and company to Chiron) as a head of oligo synthesis lab. After invention of PCR, he admitted it was a "labrious" job [6].

In May 1983, Cetus was working on Sickle Cell Anemia project. And somehow, Kary wanted to develope "a technique for easily determining the identity of the neucleotide at a give postion in a DNA molecule" [6]. Frustrated by Sanger's sequencing method, he worked out the PCR idea on his driving trip to his wooden cabin [6].

Ironically, only one consultant showed interest in Kary's idea on a Cetus annual meeting in 1984[6]. And it was not until later, by Saiki's paper [7] on Sickle Cell Anemia that the bench proof for PCR finally came.

Initially, PCR was done with Klenow fragment [7]. Its therma-unstable feature brought the tedious and error-prone trouble of adding fresh enzyme each cycle. Thanks to the discovery of Taq by Thomas D. Brock [8],

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
6. Mullis KB. The unusual origin of the polymerase chain reaction. Sci Am. 1990 Apr;262(4):56-61, 64-5. DOI:10.1038/scientificamerican0490-56 |
7. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, and Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350-4. DOI:10.1126/science.2999980 |

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