Polymerase Chain Reaction (PCR)

PCR produces billions of copies of a specific piece of DNA from trace amounts of starting material. (i.e. blood, skin cells, bone) Allows scientists to isolate pure quantities of specific DNA sequences 2 30 = over 1 billion copies of a specific DNA fragment; large enough quantity to be analyzed Used in: –Criminal Investigations to identify suspects –Sequencing the Human genome

How does DNA from the Crime Scene help in the lab?

46 Chromosomes code for 30,000 to 50,000 genes; only 5% of your DNA –Exons = DNA that is coded or expressed into proteins Noncoding DNA has more diversity; since this DNA rarely leaves the DNA to head to ribosomes –Introns = DNA that is rarely expressed –Increased number of mutations

Target DNA – the DNA that needs to be copied Primers – short pieces of DNA that are designed to attach to each end of the DNA fragment that will be replicated Taq polymerase – enzyme that reads the DNA –Comes from the bacteria Thermus aquaticus –Lives in the hot springs in Yellowstone; doesn’t fall apart in high temperatures dNTPs – 4 nucleotides with the 4 different bases that are needed to replicate DNA Buffer – gives the best environment for the enzymes to work Mg Ions – needed by DNA polymerase to make DNA copies

PCR machine is known as thermal cylcer Machine changes to three different temperature changes during one cycle Average number of cycles per run is 30 to 40

Temperature at 94ºC Denaturing temperature –The target DNA falls apart –The H bonds holding the nitrogen bases together break –2 individual strands of DNA are now present instead of a double helix.

Temperature at 65ºC Annealing Temperature –Primers attach to the ends of the Target DNA that needs to be copied –Annealing = attachment of the primers –Attach to complimentary bases of target DNA

Temperature at 72ºC Extension Temperature –Provides best temp for Taq polymerase to begin reading the DNA –Taq polymerase will synthesize a second strand of complimentary DNA –Taq polymerase always read target DNA from 3’ to 5’ end

Repeat 30 times The three temperature changes represents one cycle –Denature –Anneal –Extend Repeat 30 times 2 30 = over 1 billion copies of the Target DNA Once DNA is amplified (copied), it is visible on a gel

One Cycle has all three different temperatures.

Another Animation

PCR Animation