Polymerase Chain Reaction (PCR) NUBIO: WhY are We Different? By: Kabi Neupane, Ph.D. Edited by: Leah Spee

Polymerase 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

Discovery PCR was discovered by Kary Mullis Nobel Prize in Chemistry On a long motorcycle drive Mentally visualized the process Nobel Prize in Chemistry 1993

Properties of DNA polymearse DNA polymerase works best under optimal temperature, pH and salt concentration (as do all enzymes) PCR buffer provides optimal pH and salt condition The thermal cycler provides the proper temperatures.

Taq DNA polymerase Derived from the bacteriaThermus aquaticus Heat stable DNA polymerase Ideal temperature 72°C

Thermal Cycling A PCR machine controls the temperatures needed to amplify DNA Typical PCR go through three steps Denaturation Annealing (also called hybridization) Extension

Step 1: Denaturation Heating DNA to 94-96 °C separates the double stranded DNA Heat Cool

Step 2: Annealing Two primers are supplied. They bind to the complementary region. Typical temperature from 40 - 60 °C

What is a PRIMER? Short chains of bases that hybridize to a target piece of DNA. Primers determine the DNA fragment to be amplified in the PCR process.

Step 3: Extension Taq polymerase duplicates DNA Optimal temperature 72°C

A few PCR Applications: Genetic testing in potential parents for presence of genetic mutations that could be passed down to offspring. PCR can be used to test for HIV through detection of the actual viral genome within cells. Minute samples of DNA can be isolated from a crime scene and compared to suspects.

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