PCR Polymerase Chain Reaction

1. PCRPolymerase Chain Reaction Katherine Graf, 446L

2. History of PCR • 1953-Watson and Crick discover DNA double helix • 1957-Arthur Kornberg discovers the first DNA polymerase. • 1960’s- H. Gobind Khorana began research into the expansion of the genetic code, this led to a huge project being formed to synthesize a functional human gene. • 1969- Thomas D. Brock Thermus aquaticus is discovered, this bacteria is an extremophile that functions higher than E. coli. • 1970-Klenow, discovers a new protein of modified E. coli polymerase. • 1971-Kjell Kleppe and others propose the idea of two primer PCR.

3. Okazaki Fragments • Short strands that are formed on the lagging strand during DNA replication. Okazaki fragments are being formed in the opposite direction of the replication fork. Unlike the leading strand that uses a single primer to continue replicating, the small strand (Okazaki fragments) need to use a new primer each time.

4. Kjell Kleppe, the unknown voice of the two primer system. • In 1971 Kjell Kleppe and other researchers working in Khorana's lab, publish a paper on the importance of using two primers in PCR. This idea didn’t go anywhere. Kleppe notes that the use of a two primer system within PCR might yield specific segments of DNA.

5. History of PCR cont… • 1971- Cetus corporation in California is founded, focused on the cloning and expression of human genes. • 1976- a functioning DNA polymerase is isolated from T. aquaticus • 1977- Frederick Sanger invents his method for sequencing DNA, (Sanger Method, needs oligonucleotide primer, DNA polymerase, and modified nucleotide precursors.) • 1983- Kary Mullis proposes the idea of using two primers, thus leading to the research and development of PCR.

6. Kary Mullis, the man behind PCR • Kary Mullis, an employee of the Cetus Corporation Lab, was driving home one night when he got the inspiration to use a pair of primers as opposed to a single primer. • The use of two primers allows for one primer to direct the polymerization of the forward strand the forward strand, and another to polymerize the backward strand thus leading to exponential amplification. • The finalization of PCR was met on 16 December, 1983 when Mullis was successful in PCR amplification using two primers. • For his work, Cetus awarded Mullis a $10,000 bonus, and went on to patent PCR. • In 1993, Mullis was awarded ½ the Nobel Prize in Chemistry. Photo Courtesy of Nobel Foundation archive.

7. PCR • A 3 step sequence in which a double strand DNA is placed within a bath ranging from 94–98 °C where DNA is denatured, it breaks down into two (2) single stands. • The 2nd step the temperature drops so that the annealing of two (2) complementary primers may go forth and attach themselves to the DNA sequence that needs to be replicated. • The 3rd step is the extending step, this step employs the selected polymerase i.e. Taq. Taq will form two (2) new strands of DNA using the orginal strands as templates. • These steps all make up one (1) cycle. One must run 25-30 cycles in order to yield results of exponential DNA replication. • When running a PCR sequence, it is crucial to choose a primer that will complement the sectionof DNA that needs to be replicated.

9. Early PCR Baths “Mr. Cycle” • “Mr. Cycle” was a retrofitted machine that was used as the first PCR bath at the Cetus Corporation. This PCR bath had to be run by hand. It was effective at running PCR, however the enzyme that was being used E. Coli had to be replenished as the enzyme would break down during the denaturation step, and need to be replenished. Image courtesy of: National Museum of American History

10. Modern Thermal Cycler/PCR machines • Fortunately, there have been advancements made where the need to stand for hours on end with “Mr Cycle,” have come to an end. Modernized commercial thermal cyclers are completely automated. • Each cycler is equipped to cycle through the temperatures needed for PCR to occur.

11. Thermus aquaticus…our silent partner • 1985, two years after the discovery of PCR, Mullis and colleagues started seeking out an alternative enzyme to be used in the PCR bath. • E. coli was effective in use of PCR, but impractical as it degraded far too often. • Mullis had the idea to test the thermostable DNA polymerase from the thermophile Thermus aquaticus. • Thermus aquaticus was superior because it proved thermostable and did ntot lose activity such as the E. coli DNA polymerase.

12. Pfu DNA Polymerase • Pyrococcus furiosis (first used in lab in 1991) is a species of Archea that is capable of extreme temperatures as Taq is. It has become a contendor in the use of PCR. • Why? • Pfu has better thermostability. • Pfu is capable of going an extra step during PCR, Pfu has the ability of 3′ to 5′ exonuclease. In essence, the polymerase will work it’s way along the 5’ to 3’ strand and correct any nucleotide errors that were made whilst duplicating the DNA on the 3’ end of the DNA strand. • Because Pfu is capable of proofreading, it gives this high fidelity. • Pfu is capable of producing blunt ends as opposed to sticky ends.

13. Continuing Applications • From experimental use, PCR has become a routine procedure with many kits and thermal cyclers available. • Since the discovery of PCR, there have been many applications and widespread uses that call on PCR. • These include but are not limited to: • Gene expression, genotyping, cloning, mutagenesis, methylation analysis, sequencing, and vast use in the medical, forensic and applied sciences fields.

14. References • Kary B. Mullis – Facts. NobelPrize.org. Nobel Media AB 2018. Mon. 1 Oct 2018. https://www.nobelprize.org/prizes/chemistry/1993/mullis/facts/ • https://en.m.wikipedia.org/wiki/History_of_polymerase_chain_reaction • http://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/71-dna-structure-and-replic/okazaki-fragments.html • https://learn.genetics.utah.edu/content/labs/pcr/ • http://americanhistory.si.edu/collections/search/object/nmah_1000862

15. Questions?