POLYMERASE CHAIN POLYMERASE CHAIN REACTIONREACTION

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ObjectivesObjectives

Review DNA structure and functionReview DNA structure and function Discuss principle of PCRDiscuss principle of PCR Discover advantages and Discover advantages and

drawbacks to PCRdrawbacks to PCR Determine clinical uses for PCRDetermine clinical uses for PCR

A quick review of DNAA quick review of DNA

DNA is composed of four DNA is composed of four nucleotide bases that pair in nucleotide bases that pair in a predictable pattern.a predictable pattern.

DNA is a double stranded DNA is a double stranded molecule that forms a molecule that forms a double helix.double helix.

DNA strands are held DNA strands are held together by hydrogen bonds together by hydrogen bonds and van der Waals forces.and van der Waals forces.

Strands of DNA run Strands of DNA run antiparallel to one another antiparallel to one another with bases on the interior with bases on the interior and a phosphate sugar and a phosphate sugar backbone exterior.backbone exterior.

DNA StructureDNA Structure

PCR: Where did it come PCR: Where did it come from?from?

First developed in mid-1980’s from First developed in mid-1980’s from an idea by Kary Mullis.an idea by Kary Mullis.

Unveiled to the public in 1985.Unveiled to the public in 1985. Gained widespread popularity in Gained widespread popularity in

research circles by 1991.research circles by 1991. Mullis received the Nobel Prize for Mullis received the Nobel Prize for

Chemistry in 1993.Chemistry in 1993.

What is the principle of What is the principle of PCR?PCR?

PCR can amplify up to billions of PCR can amplify up to billions of copies of a segment of interest.copies of a segment of interest.

PCR takes advantage of DNA’s PCR takes advantage of DNA’s natural replication process.natural replication process.

The polymerase chain reaction has The polymerase chain reaction has three basic parts.three basic parts.

PCR requires several PCR requires several componentscomponents

Primers comple-Primers comple-mentary to the mentary to the flanking sequences flanking sequences of the segment of of the segment of interestinterest

Deoxynucleotide Deoxynucleotide triphosphates triphosphates (dNTPs).(dNTPs).

Template DNATemplate DNA

Taq Taq DNA DNA polymerasepolymerase

BufferBuffer A source of MgA source of Mg2+2+

ddHddH22O O A thermal cycler, A thermal cycler,

or equivalentor equivalent

How does it work?How does it work?

DS template DNA DS template DNA must be denatured.must be denatured.

Primers hybridize, or Primers hybridize, or anneal to the anneal to the template DNA.template DNA.

dNTPs are dNTPs are incorporated into the incorporated into the growing strand.growing strand.

Target DNA is Target DNA is amplified amplified exponentially.exponentially.

Benefits of PCRBenefits of PCR

The ability to amplify target DNA from very minute quantities.

Rapid identification of microorganisms that are difficult or impossible to isolate in culture.

Early diagnosis of hereditary/genetic disorders or predisposition to them. Earlier diagnosis of infectious diseases that have previously relied on immunologic techniques.

Detection of a single gene mutation.

Drawbacks to PCRDrawbacks to PCR

PCR reaction mixtures can be easily PCR reaction mixtures can be easily contaminated.contaminated.

Some consumables used in the Some consumables used in the process can be expensive.process can be expensive.

Requires the use of special Requires the use of special equipment.equipment.

Users may need new/refresher Users may need new/refresher training.training.

Clinical Uses of PCRClinical Uses of PCR

Early detection of HIVEarly detection of HIV Identification of Identification of M. M.

tuberculosis tuberculosis without without cultureculture

Detection of bacterial Detection of bacterial DNA in middle ear DNA in middle ear fluid of childrenfluid of children

Lyme disease Lyme disease diagnosis from joint diagnosis from joint fluidfluid

Gonorrhea, Gonorrhea, Herpes, and Herpes, and ChlamydiaChlamydia detection from detection from genital swabgenital swab

Rapid detection of Rapid detection of Helicobacter pyloriHelicobacter pylori

Rapid detection of Rapid detection of MRSAMRSA