AMPLIFICATION OF DNA BY THE

POLYMERASE CHAIN REACTION

PCR The polymerase chain reaction is a

process that allows individual DNA fragments to be propagated in bacteria and isolated in large amounts

The DNA polymerases used in PCR reactions are heat-stable enzymes from bacteria such as Thermus aquaticus: Taq polymerase

BACKGROUND First developed in mid-1980’s from an

idea by Kary Mullis. Unveiled to the public in 1985. Gained widespread popularity in

research circles by 1991. Mullis received the Nobel Prize for

Chemistry in 1993

COMPONENTS NEEDED Primers comple-

mentary to the flanking sequences of the segment of interest

Deoxynucleotide triphosphates (dNTPs).

Template DNA

Taq DNA polymerase

Buffer A source of Mg2+

ddH2O A thermal cycler,

or equivalent

GENERAL PROCESS DS template DNA must be denatured

at 95C Primers hybridize, or anneal, to the

template DNA dNTPs are incorporated into the

growing strand Basically cell free DNA replication Process of heating and cooling is

repeated many times to make possibly billions of copies

Each copy serves as a template for a new strand in the next “generation”

Target DNA is amplified exponentially

THOUGHTS TO CONSIDER If enough of gene sequence is known, PCR

provides powerful method for detecting small amounts of specific DNA molecules in a complex mixture of other molecules Need to know enough for specific primers to be

made

Used in medicine, forensic investigations, research and development, recombinant DNA techniques

PCR reactions may be easily contaminated, aseptic technique critical

Disclaimer This workforce solution was funded by a grant

awarded under the President’s Community-Based Job Training Grants as implemented by the U.S. Department of Labor’s Employment and Training Administration.  The solution was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor.  The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not limited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership.  This solution is copyrighted by the institution that created it.  Internal use by an organization and/or personal use by an individual for non-commercial purposes is permissible.  All other uses require the prior authorization of the copyright owner.