Polymerase Chain Reaction

Polymerase Chain Reaction

Mrs. StewartMedical Interventions

Polymerase Chain Reaction

a lab technique that produces numerous copies of a specific segment of our DNA in a relatively short period of time. three-step processrepeated over and overproduces identical copies of the target

sequence.

Kary Mullis1983 – Mullis

and his colleagues invented the PCR technique

Nobel Prize in 1993 

PCR components

DNA (Taq) polymeras

e

Primers Nucleotides

Target DNA

sequence

Taq PolymeraseThe most widely used polymerase is

that from Thermus aquaticus (Taq) – Thermophilic bacteria

Thermophilic bacterium lives in hot springs and capable of growing at 70 -75 C

3 steps in a PCR1.Denature2.Anneal3.Extension

DenatureThe DNA is heated to 95oC, causing

the double stranded DNA to denature by breaking the hydrogen bonds between the strands.

Denaturation

AnnealThe temperature of the sample is

lowered to between 32-72oC, causing the primers to hybridize or "anneal" to their complementary sequences on either side of the target sequence.

Anneal

ExtensionThe temperature of the sample is

heated to between 72-75oC, which is the optimal temperature for the Taq polymerase enzyme to function.

Taq polymerase binds and extends a complementary DNA strand from each primer (adding approximately 60 bases per second, using the free-floating nucleotides)

Extension

As amplification proceeds, the DNA sequence between primers doubles after each cycles

(The amplification of the target sequence proceeding in an exponential fashion ( 1 2 4 8 16................) up to million of times the starting amount until enough is present to be seen by gel electrophoresis.

How many cycles?Most PCRs should include only 25 –

35 cycles. Depends on the amount of

starting material

Advantages of PCRUseful, non-invasive procedureSimplicity of the procedureSensitivity of the PCR

Disadvantages of PCRFalse positive results (cross contamination). False negative results (e.g. rare of

circulating fetal cells).