POLYMERASE CHAIN REACTION

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Objectives b Review DNA structure and function b Discuss principle of PCR b Discover advantages and drawbacks to PCR b Determine clinical uses for PCR

A quick review of DNA b DNA is composed of four nucleotide bases that pair in a predictable pattern. b DNA is a double stranded molecule that forms a double helix. b DNA strands are held together by hydrogen bonds and van der Waals forces. b Strands of DNA run antiparallel to one another with bases on the interior and a phosphate sugar backbone exterior.

DNA Structure

PCR: Where did it come from? b First developed in mid-1980’s from an idea by Kary Mullis. b Unveiled to the public in b Gained widespread popularity in research circles by b Mullis received the Nobel Prize for Chemistry in 1993.

What is the principle of PCR? b PCR can amplify up to billions of copies of a segment of interest. b PCR takes advantage of DNA’s natural replication process. b The polymerase chain reaction has three basic parts.

PCR requires several components b Primers comple- mentary to the flanking sequences of the segment of interest b Deoxynucleotide triphosphates (dNTPs). b Template DNA b Taq DNA polymerase b Buffer b A source of Mg 2+ b ddH 2 O b A thermal cycler, or equivalent

How does it work? b DS template DNA must be denatured. b Primers hybridize, or anneal to the template DNA. b dNTPs are incorporated into the growing strand. b Target DNA is amplified exponentially.

Benefits of PCR b b The ability to amplify target DNA from very minute quantities. b b Rapid identification of microorganisms that are difficult or impossible to isolate in culture. b Early diagnosis of hereditary/genetic disorders or predisposition to them. Earlier diagnosis of infectious diseases that have previously relied on immunologic techniques. b Detection of a single gene mutation.

Drawbacks to PCR b PCR reaction mixtures can be easily contaminated. b Some consumables used in the process can be expensive. b Requires the use of special equipment. b Users may need new/refresher training.

Clinical Uses of PCR b Early detection of HIV b Identification of M. tuberculosis without culture b Detection of bacterial DNA in middle ear fluid of children b Lyme disease diagnosis from joint fluid b Gonorrhea, Herpes, and Chlamydia detection from genital swab b Rapid detection of Helicobacter pylori b Rapid detection of MRSA