Polymerase Chain Reaction (PCR) is a method for producing an extremely
large number of copies of a specific DNA sequence through amplification.
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.
polymerse is from the thermophilic bacterium
, which was discovered by Thomas Brock in the 1960s
in the hot springs of
Yellowstone National Park
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.
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
= 2(A + T) + 4(G + C), where A, T, G, and C represent the
numbers of each nucleotide in the primer sequence.
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
in Chemistry in 1993 for his PCR method.
- 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
- 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.
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.
Newton, R.C., & Graham, A. (1997). PCR. (2nd ed.). New York: BIOS
Russell, P. J. (2000). Fundamentals of genetics. (2nd ed.). San Francisco,
CA: Addison Wesley