PCR and Forensics

1. PCR and Forensics Tina Doss Applied Biosystems

2. Directory for Forensic DNA Presentation Biology Review Nomenclature for DNA Markers Forensic DNA – Overview Sample Collection PCR References

3. Forensic DNA – Biology Review DNA has two primary purposes: • To make copies of itself so cells can divide and carry on the same information. • To carry instructions on how to make proteins.

4. Forensic DNA – Biology Review DNA has 3 parts: • A base • A sugar (pentose) • A phosphate group Click picture for DNA Structure animation

5. Forensic DNA – Biology Review • A DNA sequence is normally written and read from 5’ to 3’. • DNA Polymerase is the enzyme that will “write” sequences. • Much like we read in a certain direction, like left to right. Click picture for DNA Replication animation

6. Forensic DNA – Biology Review • DNA has 2 strands linked through process called hybridization. • Complementary base pairing is completed through hydrogen bonds between bases: • A=T • G=C • The strands run anti-parallel _ Click picture for Replication Fork animation

7. Forensic DNA – Biology Review • DNA may be denatured (split 2 strands) by: • Heat DNA to near boiling temperatures • Place DNA in a salt solution of low ionic strength • Expose DNA to chemical denaturants (ex: Urea)

8. Forensic DNA – Biology Review • Denaturation is reversible. • Process of 2 complementary DNA strands coming back together is called renaturation or reannealing. • In lab, renaturation occurs during cold cycles.

9. Forensic DNA – Biology Review • Approximately 3 billion base pairs in a single copy of human genome • Chromosome = dense packet of DNA wrapped around proteins called histones

10. Forensic DNA – Biology Review • Somatic (body) cells are diploid = 2 sets of each chromosome • 23 pairs or 46 chromosomes total • 22 pairs of autosomes and 1 pair ofsex chromosomes

11. Forensic DNA – Biology Review • Genes have exons (protein coding portions) and introns (non coding portions). • Markers used for human identity testing are found in introns either between genes or within genes.

12. Forensic DNA – Biology Review • Location of a gene or DNA marker is called a locus. • Thousands of loci have been characterized and mapped to particular regions of chromosomes thanks to Human Genome Project.

13. p (short arm) Centromere q (long arm) Telomere Telomere Forensic DNA – Biology Review • Designating Physical Chromosome Locations: Band 2 on p-arm

14. Write as much information as you can about DNA and the biology review. _____________ is like ____________ because: Four Squares Questions or Confusions? Applications for use or relationship to what I know now.

15. Forensic DNA – Nomenclature for DNA Markers • If marker is part of a gene or falls within a gene – the gene name is used • EX: STR TH01-11 • TH = human tyrosine hydroxylase gene • 01 = means the repeat region is located within intron 1 of the tyrosine hydroxylase gene • 11 = located on chromosome 11

16. Forensic DNA – Nomenclature for DNA Markers • If marker is outside gene regions designated by chromosomal position the naming is slightly different: • EX: D5S18 • D = DNA • 5 = Chromosome # • S = DNA marker is a single copy in the genome • 18 = indicates the historical order in which the marker was discovered

17. Forensic DNA – Nomenclature for DNA Markers • So what does D1S80 stand for? • EX: D1S80 • D = DNA • 1 = Chromosome # • S = DNA marker is a single copy in the genome • 80 = indicates the order in which the marker was discovered • Where would this type of marker be found – within the gene regions or outside the gene regions? Outside the gene regions based on the fact that it does not bear the name of the gene it is located in.

18. Sample Collection Purification Quantification Forensic DNA - Overview STR PCR

19. Forensic DNA – Sample Collection Sources of biological materials used for PCR-based DNA typing: • Blood and Blood Stains • Semen and Semen Stains • Bones and Teeth • Hair (Root and Shaft) • Saliva • Urine and Feces • Debris from Fingernails • Cigarette Butts • Postage Stamps • Envelope Sealing Flaps • Dandruff • Fingerprints

20. Forensic DNA – PCR • PCR = Polymerase Chain Reaction • Process in which a specific region of DNA is replicated over and over again • VNTR = VariableNumber of TandemRepeats • STR = Short Tandem Repeats

21. Forensic DNA – PCR • The Power of PCR: • Only need a small amount of DNA. • Lab and analysis is completed quickly. • May use your own DNA as template.

22. Forensic DNA – PCR • Need 2 primers to “flank” region of DNA to be copied. • Use a forward and reverse primer to start as the starting point and isolate the target DNA sequence.

23. Forensic DNA – PCR • Also need polymerase that can build bases in the correct order from template DNA strand. • MgCl2 is needed for activation of the polymerase.

24. Forensic DNA – PCR In the Master mix: • Buffer – stabilizes pH for Polymerase to work • MgCl2 for DNA Polymerase activation • dNTP (Deoxynucleoside tri-phosphate) • These are your A, T, G and C’s • AmpliTaq Gold = DNA Polymerase

25. Positive Control Valuable indicator of whether or not any of the PCR components failed or were not added to the experiment. Also valuable to detect if thermal cycling parameters are working for amplification of DNA. DNA template is amplified with the same primers. Negative Control This is the entire PCR reaction mixture without any DNA template. (Use water or buffer instead of DNA) Useful to assess whether or not PCR components are contaminated by DNA. Forensic DNA – PCR

26. 32 CYCLES 1 HOLD 2 HOLDS 95oC 95oC 72oC 72oC 10:00 0:15 65oC 0:40 10:00 0:30 4oC ∞ Activation During this stage, AmpliTaq Gold DNA polymerase is activated. The heat causes the pH of the buffer to drop and the chemical modification on the polymerase to fall off, activating the enzyme. Forensic DNA – PCR • The instrument that heats and cools a DNA sample for PCR is called a Thermal Cycler. Thermal Cycling Parameters:

27. 32 CYCLES 1 HOLD 2 HOLDS 95oC 95oC 72oC 72oC 10:00 0:15 65oC 0:40 10:00 0:30 4oC ∞ Denature Stage Activation Heat is used instead of helicase to unwind the DNA strand. The DNA strand is pulled apart. Forensic DNA – PCR Thermal Cycling Parameters:

28. 32 CYCLES 1 HOLD 2 HOLDS 95oC 95oC 72oC 72oC 10:00 0:15 65oC 0:40 10:00 0:30 4oC ∞ Denature Activation Annealing This is the stage where your forward and reverse primers attach to the DNA strand. Remember that the primers are looking for the complimentary bases and will target on piece of DNA to amplify. Forensic DNA – PCR Thermal Cycling Parameters:

29. 32 CYCLES 1 HOLD 2 HOLDS 95oC 95oC 72oC 72oC 10:00 0:15 65oC 0:40 10:00 0:30 4oC ∞ Denature Annealing Activation Extension During this stage, polymerase attaches and copies the bases from the parental DNA strand in the 5’ to 3’ direction. Forensic DNA – PCR Thermal Cycling Parameters:

30. The denature, anneal, and extension stages repeat as many as 40 and as few as 30 times. For D1S80, the cycle repeats 32 times. 32 CYCLES 1 HOLD 2 HOLDS 95oC 95oC 72oC 72oC 10:00 0:15 65oC 0:40 10:00 0:30 4oC ∞ Forensic DNA – PCR Thermal Cycling Parameters:

31. 32 CYCLES 1 HOLD 2 HOLDS 95oC 95oC 72oC 72oC 10:00 0:15 65oC 0:40 10:00 0:30 4oC ∞ Denature Annealing Extension Activation Final Extension This stage is to allow sufficient time for all DNA fragments from previous cycles to finish extension. Forensic DNA – PCR Thermal Cycling Parameters:

32. 32 CYCLES 1 HOLD 2 HOLDS 95oC 95oC 72oC 72oC 10:00 0:15 65oC 0:40 10:00 0:30 4oC ∞ Denature Annealing Extension Final Extension Activation Final Hold This stage is to slow down all the processes and help keep the solution stable. This is like putting your sample in the refrigerator. Forensic DNA – PCR Thermal Cycling Parameters:

33. Forensic DNA – PCR Thermal Cycling Parameters: • Now it’s your turn. • Label the following stages on the cycling protocol below: • Annealing stage, Denature stage, Extension Stage, Final Extension Stage, Activation Stage, Final Hold Stage. • Make sure to add a one sentence summary for each stage.

34. Final Extension Annealing Stage Denaturing Stage Activation Stage Extension Stage Final Hold Forensic DNA – PCR Thermal Cycling Parameters Answers:

35. Write as much information as you can about PCR relates to DNA. Summarize PCR: Four Squares Questions or Confusions? Applications for use or relationship to what I know now.

36. Forensic DNA - References • Butler, John M. (2005). Forensic DNA Typing: Biology and Technology Behind STR Markers. Academic Press. • Daugherty, Ellyn (2006). Biotechnology: Science for the New Millenium. St. Paul: Paradiigm Publishing. • Furtado, Manohar (2007). Lead Scientist, Foster City, Applied Biosystems. • Fang, Rixun (2007). Lead Scientist, Foster City, Applied Biosystems. • Vatta, Paolo (2007). Lead Scientist, Foster City, Applied Biosystems.