Polymerase Chain Reaction Polymerase Chain Reaction (PCR)(PCR)

NUBIO: WhY are We NUBIO: WhY are We Different?Different?

By: Kabi Neupane, Ph.D.By: Kabi Neupane, Ph.D.Edited by: Leah SpeeEdited by: Leah Spee

Polymerase Chain ReactionPolymerase Chain Reaction

Polymerase: DNA polymerase– DNA polymerase is an enzyme that

duplicates DNA during interphase of the cell cycle.

Chain Reaction: The product of a reaction is used to amplify the same reaction – Results in rapid increase in the

product

DiscoveryDiscovery

PCR was discovered by Kary PCR was discovered by Kary MullisMullis– On a long motorcycle driveOn a long motorcycle drive– Mentally visualized the processMentally visualized the process

Nobel Prize in ChemistryNobel Prize in Chemistry– 19931993

Properties of DNA Properties of DNA polymearse polymearse

DNA polymerase works best under DNA polymerase works best under optimal temperature, pH and salt optimal temperature, pH and salt concentration (as do all enzymes)concentration (as do all enzymes)

– PCR buffer provides optimal pH and PCR buffer provides optimal pH and salt conditionsalt condition

– The thermal cycler provides the The thermal cycler provides the proper temperatures.proper temperatures.

Taq DNA polymeraseTaq DNA polymerase

Derived from the Derived from the bacteriabacteriaThermus aquaticusThermus aquaticus

Heat stable DNA polymeraseHeat stable DNA polymerase Ideal temperature 72°CIdeal temperature 72°C

Thermal CyclingThermal Cycling

A PCR machine controls the A PCR machine controls the temperatures needed to amplify temperatures needed to amplify DNADNA

Typical PCR go through three stepsTypical PCR go through three steps– DenaturationDenaturation– Annealing (also called hybridization)Annealing (also called hybridization)– ExtensionExtension

Step 1: DenaturationStep 1: Denaturation

Heating DNA to 94-96 °CHeating DNA to 94-96 °C

separates the double separates the double

stranded DNAstranded DNAHeat Cool

Step 2: Step 2: AnnealingAnnealing Two primers are supplied.Two primers are supplied. They bind to the complementary They bind to the complementary

region.region. Typical temperature from 40 - 60 °CTypical temperature from 40 - 60 °C

What is a PRIMER?What is a PRIMER?

Short chains of bases that Short chains of bases that hybridize to a target piece of hybridize to a target piece of DNA.DNA.

Primers determine the DNA Primers determine the DNA fragment to be amplified in the fragment to be amplified in the PCR process.PCR process.

Step 3: ExtensionStep 3: Extension

Taq polymerase duplicates DNATaq polymerase duplicates DNA Optimal temperature 72°COptimal temperature 72°C

A few PCR Applications: A few PCR Applications:

Genetic testing in potential parents for Genetic testing in potential parents for presence of genetic mutations that presence of genetic mutations that could be passed down to offspring.could be passed down to offspring.

PCR can be used to test for HIV PCR can be used to test for HIV through detection of the actual viral through detection of the actual viral genome within cells.genome within cells.

Minute samples of DNA can be isolated Minute samples of DNA can be isolated from a crime scene and compared to from a crime scene and compared to suspects. suspects.

PCR AnimationPCR Animation

http://www.dnalc.org/resources/animations/pcr.html