2. DNA STRUCTURE 5’ end3’ end 5’ end3’ end Guanine Adenine Thymine Cytosine * Deoxyribose Sugar * * Phosphate * * Nitrogen Base * = “Nucleotide” “Purines” “Pyrimidines”

3. Polymerase Chain Reaction Makes amplification of very small samples of DNA possible Technique is indispensible in medical testing and forensics along with other fields.

4. In the 1960s Thomas Strock discovered Thermus aquaticus (aka “Taq”) Molecular Biologist, Kary Mullis, a North Carolina native developed the technique in 1983 while. In 1993, he was awarded the Nobel Prize in Chemistry for this achievement. Development of PCR

5. How DNA Is Collected Urine Semen Saliva Bone Teeth Blood Tissue Hair

7. What Is Needed for PCR? Template (the DNA you want to amplify for the study) Sequence-specific primers flanking the target sequence: Nucleotides (dATP, dCTP, dGTP, dTTP) Magnesium ions (enzyme cofactor) Buffer, containing salt Taq polymerase 3’ 5’ 3’ 5’ 3’ 5’ Forward primer Reverse primer Target sequence

8. How Does PCR Work? Heat (94°C) to denature DNA strands Cool (60°C) to anneal primers to template Warm (72°C) to activate Taq polymerase, which extends primers and replicates DNA Repeat multiple cycles

9. Denaturing Template DNA 3’ 5’ 3’ 5’ 3’ 5’ Heat causes DNA strands to separate Denaturation of DNA at 94°C

10. Annealing Primers 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Primers bind to the template sequence Taq polymerase binds to double-stranded substrate Primers anneal at 60°C

11. Taq Polymerase Extends… Taq polymerase extends primer DNA is replicated Extends at 72°C 3’ 5’ 3’ 5’ 3’5’ 3’ 5’ 3’ 5’ 3’5’ 3’ 5’ 3’

12. Exact-length Target Product is Made in the Third Cycle 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ Cycle 1 Cycle 2 Cycle 3

13. Milestones in Forensic DNA Analysis 1985 Alex Jeffreys develops RFLP. 1990 PCR analysis using single locus STR begins. 1992 FBI initiates STR work. 1994 DNA Identification Act provides funding for national DNA database. 1995 OJ Simpson trial focuses public attention on DNA evidence. 1998 FBI starts CODIS database: All remains from Swissair disaster were identified using STR DNA profiling. 2001 Many remains from the World Trade Center disaster in New York are identified using a combination of DNA profiling approaches. 2004 California’s Proposition 69 provides funding to maintain a DNA database. 2004 Indian Ocean tsunami: Interpol and other world agencies to use DNA profiling to identify victims.

14. Special Considerations for PCR in Forensics PCR amplifies ALL DNA, especially susceptible to contamination –DNA from person doing the collection –DNA from lab tech –Residual DNA on lab equipment –Contaminated reagents ALWAYS RUN A NEGATIVE CONTROL!!!!