Polymerase Chain Reaction

1. Polymerase Chain Reaction PCR (PCR)

2. 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 • 230 = over 1 billion copies of a specific DNA fragment; large enough quantity to be analyzed

3. What DNA is Used? • 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

4. What ingredients are needed? • 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

5. PCR: How do we get started?

6. How Does PCR Work? • PCR machine is known as thermal cylcer • Machine changes to three different temperatures during one cycle • Average number of cycles per run is 30 to 40 What happens at each temperature change?

7. Denaturing Temperature • Temperature at 94°C • 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.

8. Temp between 56-65 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 Annealing Temperature

9. Temperature at 72°C 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 Extension Temperature

10. Repeat 30 times • The three temperature changes represents one cycle • Denature • Anneal • Extend • Repeat 30 times 230 = over 1 billion copies of the Target DNA • Once DNA is amplified (copied), it is visible on a gel

11. PCR Animation

12. By the 4th Cycle = 32 Copies

13. D stands for the chromosome, and the S stands for map location of the chromosome, and the 80 is the locus point D1S80 • Locus is on chromosome 1 • Intron – noncoding region of Chromosome 1 • Each person has two copies of D1S80, one from each parent • VNTR – Variable Number of Tandem Repeats • Consists of a repeating 16 base pattern (10 repeats to >40 repeats) • Depending on how many repeating patterns present, determines the size of your D1S80 locus

14. Homozygous or Heterozygous

15. Determining your genotype!

16. Locus with variability D1S80

17. Huntington’s Chorea • Found on Chromosome 4 • Noncoding region that actually causes genetic disease • People with Huntington’s have a section on chromosome 4 that has 35 or more of three base repeating pattern CAG (trinucleotide repeat) • CAG normally codes for glutamine • Huntington’s patients will have a long line of glutamine produced

18. Effects of Huntingtons

19. Dominant Autosomal Disease