Polymerase Chain Reaction

Julia Adams
Research in Molecular Genetics
Technique of the Week
December 14, 2002

Definition
Reagents
Taq Polymerase
PCR Cycle
Kary Mullis
PCR Applications
References

Definition

Polymerase Chain Reaction (PCR) is a method for producing an extremely large number of copies of a specific DNA sequence through amplification.

Reagents

In addition to the template DNA you want to amplify, you need two oligonucleotide primers.  These primers are single-standed and contain the 5' and 3' sequences (20-30 nucleotides long) that flank your desired DNA.  These primers are made synthetically and are usually diluted to a 20 µM working solution for a 1µM dilution in the reaction mixture.  Deoxynucleoside triphosphates (dNTPs) are also needed for building new DNA.  PCR is usally performed with a 100 µM concentration of dNTPs, and Taq polymerase has higher accuracy at lower concentrations.  Magnesium chloride (MgCl2) is also essential for dNTP incorporation.  The optimum concentration range for MgCl2 is 1.0-1.5 mM; low Mg2+ leads to low PCR yields and an excess results in nonspecific products.  Finally, Taq DNA polymerase is needed to polymerize the new DNA.

Taq Polymerase

Taq polymerse is from the thermophilic bacterium Thermus aquaticus, which was discovered by Thomas Brock in the 1960s in the hot springs of Yellowstone National ParkTaq replicates DNA at 74°C by catalyzing polymerization in the 5' to 3' direction at a rate of 35–100 nucleotides per second.  It also possesses 5' to 3' exonuclease activity as a proofreading mechanism.

PCR Cycle

The entire PCR cycle takes place in a thermocycler and is as follows:
  • Denaturation of DNA (90-94°C)
  • Annealing of primers (45-70°C)
  • Extension by polymerase (70-75°C)
  • Repeat process 20-30 times
The annealing temperature will vary depending on how well the primers match the desired sequence - the more perfect the match the higher the temperature.  A common equation used for the determination of annealing temperature is T m = 2(A + T) + 4(G + C), where A, T, G, and C represent the numbers of each nucleotide in the primer sequence.

Kary Mullis

Kary Mullis was working at Cetus Corporation in Berkeley doing DNA synthesis and finding point mutations with oligonucleotides and radioactively labeled dNTPs when he came up with the idea for PCR.  He won the Nobel Prize in Chemistry in 1993 for his PCR method.

PCR Applications
  • Amplification of DNA for cloning - Add linkers with restriction sites to primers, anneal at a lower temperature initially, insert PCR product into a cloning vector.
  • Disease diagnosis (eg. HIV testing) - Amplify HIV-specific DNA from blood instead of performing an ELISA or western blot, which look for antibodies against HIV.
  • Sex determination (eg. cattle embryos) - One cell can be removed from an embryo at an early stage of development (8 cells or more) for amplification of a Y chromosome gene such as the sry gene, which encodes a testis-specific transcription factor.
  • Molecular evolution
    • Scientists have amplified a hypervariable region of Neanderthal mitochodrial DNA and compared it to human DNA, leading them to conclude that Neanderthals and modern humans developed separately and are therefore separate species.
    • Macalester College students have been working on developing a DNA-based taxonomic key of the freshewater muscle species of the St. Croix River using PCR to amplify an internal transcribed spacer.
  • Forensics - PCR is used to amplify segments with variable numbers of tandem repeats (VNTRs), and the frequencies of these segments in a population are used to determine the probability of finding an individual with the same DNA profile.

References

Bourgaize, D., Jewell, T. R., & Buiser, R. G. (2000). Biotechnology: Demystifying the concepts.
   San Francisco, CA: Addison Wesley Longman, Inc.

Krings, M., Stone, A., Schmitz, R. W., Krainitzki, H., Stoneking, M., & Pääbo, S. (1997).
     Neandertal DNA sequences and the origin of modern humans. Cell, 90 (1), 19-30.

Mahbubani, M. H., & Bej, A. K. (1994). Applications of polymerase chain reaction methodology
   in clinical diagnostics. In H. G. Griffin & A. M. Griffin (Eds.). (1994). PCR technology: Current
    innovations (pp. 307-326). Ann Arbor, MI: CRC Press, Inc.

Montgomery, M. K. (2001, Nov. 28). Cloning and manipulation of DNA III. Lecture presented at
   Macalester College, St. Paul, MN.

Mullis, K. B. (2002). Kary B. Mullis—Nobel Lecture. Nobel e-Museum. <http://www.nobel.se/
   chemistry/laureates/1993/mullis-lecture.html> (2002, Sept. 14).

Newton, R.C., & Graham, A. (1997). PCR. (2nd ed.). New York: BIOS Scientific Publishers
   Limited.

Russell, P. J. (2000). Fundamentals of genetics. (2nd ed.). San Francisco, CA: Addison Wesley
   Longman, Inc.