Polymerase Chain Reaction

1. DNA Fingerprinting Polymerase Chain Reaction

2. What is DNA profiling? DNA profiling is the use of molecular genetic methods to determine the exact genotype of a DNA sample to distinguish one human being from another . The unique genotype of each sample is called a DNA profile.

3. How do crime scene investigators create a DNA profile? 1. Evidence is collected at the crime scene: Blood Tissue Semen Urine Hair Teeth Saliva Bone

4. 2. DNA is extracted from sources at the crime scene and from victim and suspects How do crime scene investigators create a DNA profile?

5. Crime Scene Investigators search in areas of the genome that are unique from individual to individual and are “anonymous” (control no known trait or function). The areas examined are Short Tandem Repeats or STR’s. Since humans are 99.9% identical where do crime scene investigators look for differences in DNA profiles?

6. 5’ 3’ Starting DNA Template 3’ 5’ To determine the genotype (DNA profile) Crime Scene Investigators make billions of copies of the target sequence using PCR. Target DNA PCR

7. What’s the point of PCR? • PCR, or polymerase chain reaction, makes copies of a specific piece of DNA • PCR allows you to look at one specific piece of DNA by making copies of onlythat piece of DNA • PCR is like looking for a needle in a haystack, and then making a haystack out of the needle

8. PCR: Three Main Steps • 1. Denature—DNA unzips • Hottest part of the cycle • 2. Anneal—primers grab on • Coolest part of the cycle • 3. Extending/synthesis—polymerase does its thing • Repeat: After the extend part of the cycle, return to denature

9. PCR: Three Main Steps http://www.youtube.com/watch?v=_YgXcJ4n-kQ

10. Materials needed for a PCR reaction DNA of interest—crime scene DNA? DNA of a missing person? Primers—attached to the area you want to copy , gives polymerase a place to start.

11. Materials needed for a PCR reaction Taq polymerase —attaches to primers to extend the DNA. This is the enzyme that takes nucleotides and adds them to the complementary strand to copy it. dNTP’s —nucleotides that are added to the single strand of DNA in order to copy it. They are A’s, T’s, G’s and C’s floating around in the reaction. They are grabbed and added when needed.

12. What is needed for PCR? Reverse primer 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ Forward primer Target sequence The PCR ReactionWhat do you need? • Template (containing the STR you want to amplify for the study) • Sequence-specific primers flanking the target sequence • Nucleotides (dATP, dCTP, dGTP, dTTP) • Magnesium chloride (enzyme cofactor) • Buffer, containing salt • Taq polymerase

13. Taq polymerase • Polymerases are enzymes that add nucleotides to the free 3’ end of DNA. • Taqpolyermase is thermostable (stable at high temperature 50-80 degrees Celsius). • Comes from Thermusaquaticus, a bacterium found in hot springs. • T. aquaticus was discovered in a geyser at Yellowstone park.

15. Heat (94oC) to denature DNA strands Cool (52oC) to anneal primers to template Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA Repeat 35 cycles The PCR ReactionHow does it work?

16. PCR Temperatures vs. Steps

17. What is happening in the PCR tube while in the thermocycler? PCR Animation http://www.bio-rad.com/flash/07-0335/07-0335_PCR.html

18. Crime Scene Investigator PCR Basics™ProceduresOverview

19. PCR Possible Problems • High risk of contamination • Can copy degraded DNA samples • Use gloves during entire lab!!! • DNases on your skin can degrade the DNA samples • Optimization—determining the best conditions for the reaction can be difficult