1. Tina Doss Applied Biosystems
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)
-Chemical denaturants like urea and formamide form hydrogren bonds with the nucleotides preventing association with complementary DNA Strand.

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.
-Chemical denaturants like urea and formamide form hydrogren bonds with the nucleotides preventing association with complementary DNA Strand.

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 of sex 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.
-Genes make up about 5% of the human genomic DNA
-Non-protein coding regions of DNA make-up the rest of the chromosomal material.
-B/c not directly related to making proteins they are sometimes referred to as “junk” DNA
-Polymorphic (variable) markers that differ among individuals can be found throughout the non-coding region of human genome.

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.
-Genes make up about 5% of the human genomic DNA
-Non-protein coding regions of DNA make-up the rest of the chromosomal material.
-B/c not directly related to making proteins they are sometimes referred to as “junk” DNA

13. Forensic DNA – Biology Review
Designating Physical Chromosome Locations:
Telomere
p (short arm)
Band 2 on p-arm
Centromere
q (long arm)
-Telomere are the tips of the chromosome
-Bands are counted from the centromere out.
Telomere

14. Four Squares
Write as much information as you can about DNA and the biology review.
_____________ is like ____________ because:
Questions or Confusions?
Applications for use or relationship to what I know now.
-Have students copy this down on the next blank page in their notebooks. Give them 5 minutes to fill it out quietly by themselves.
-After 5 minutes, allow them to partner up and share their answers. Have students write down items they may have missed by themselves.
-Do a group share. Call on a couple of groups. Have one student say one item their partner wrote and vice versa.

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
STR = Short Tandem Repeat

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?
-This slide is a Q-A to go over with the class to help make sure everyone understands what they have learned so far on nomenclature.
-D1S80 is the name of the piece of DNA students will separate in the lab following this Ppt.
Outside the gene regions based on the fact that it does not bear the name of the gene it is located in.

18. Forensic DNA - Overview
Sample Collection
Purification
Quantification
STR PCR
-Need to be very careful when collecting samples. Professionals are trained on proper techniques.
-The DNA is then purified out using a series of techniques with either silicon beads or magnetic beads.
-Quantification is done to determine how much DNA was purified out of the sample. Ideally, to identify a person, the DNA should be at about 1ng/10 uL. After the quantification step, dilute the sample down to the proper amount. Then run an identification PCR lab (16 different locuses run at the same time).

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 = Variable Number of Tandem Repeats
STR = Short Tandem Repeats
STR = Short Tandem Repeat
-For D1S80, the repeat segment of DNA is 16 bp long. It is repeated next to one another totally adjacent. There may be as few as 14 repeats and as many as 41 repeats on Chromosome 1. This is how we can identify an individual.
-Usually 16 different loci – like D1S80 – are used to accurately identify an individual to 1 in over 10 billion.

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.
-polymerase is the enzyme that matches A with T and G with C.
-Most common polymerase used in lab is Taq Polymerase, which is the enzyme that comes from a bacterium named Thermus Aquaticus that inhabits hot springs.

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. Forensic DNA – PCR Positive Control Negative 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.
-polymerase is the enzyme that matches A with T and G with C.
-Most common polymerase used in lab is Taq Polymerase, which is the enzyme that comes from a bacterium named Thermus Aquaticus that inhabits hot springs.

26. Forensic DNA – PCR
The instrument that heats and cools a DNA sample for PCR is called a Thermal Cycler.
Thermal Cycling Parameters:
95oC
10:00
72oC
65oC
0:15
0:30
0:40
4oC ∞
1 HOLD
32 CYCLES
2 HOLDS
Activation
-Most common Thermal Cycler is the GeneAmp® PCR System 9600 from Applied Biosystems.
-The 9600 can heat and cool 96 samples in an 8 x 12-well microplate format at a rate of approximately 1oC per second.
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.

27. Forensic DNA – PCR ∞ Thermal Cycling Parameters: 95oC 10:00 72oC 65oC
0:15
0:30
0:40
4oC ∞
1 HOLD
32 CYCLES
2 HOLDS
Activation
Denature Stage
-Most common Thermal Cycler is the GeneAmp® PCR System 9600 from Applied Biosystems.
-The 9600 can heat and cool 96 samples in an 8 x 12-well microplate format at a rate of approximately 1oC per second.
Heat is used instead of helicase to unwind the DNA strand. The DNA strand is pulled apart.

28. Forensic DNA – PCR ∞ Thermal Cycling Parameters: 95oC 10:00 72oC 65oC
0:15
0:30
0:40
4oC ∞
1 HOLD
32 CYCLES
2 HOLDS
Activation
Denature
Annealing
-Most common Thermal Cycler is the GeneAmp® PCR System 9600 from Applied Biosystems.
-The 9600 can heat and cool 96 samples in an 8 x 12-well microplate format at a rate of approximately 1oC per second.
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.

29. Forensic DNA – PCR ∞ Thermal Cycling Parameters: 95oC 10:00 72oC 65oC
0:15
0:30
0:40
4oC ∞
1 HOLD
32 CYCLES
2 HOLDS
Activation
Denature
Extension
Annealing
-Most common Thermal Cycler is the GeneAmp® PCR System 9600 from Applied Biosystems.
-The 9600 can heat and cool 96 samples in an 8 x 12-well microplate format at a rate of approximately 1oC per second.
During this stage, polymerase attaches and copies the bases from the parental DNA strand in the 5’ to 3’ direction.

30. Forensic DNA – PCR ∞ Thermal Cycling Parameters:
The denature, anneal, and extension stages repeat as many as 40 and as few as 30 times. For D1S80, the cycle repeats 32 times.
95oC
10:00
72oC
65oC
0:15
0:30
0:40
4oC ∞
1 HOLD
32 CYCLES
2 HOLDS
-Most common Thermal Cycler is the GeneAmp® PCR System 9600 from Applied Biosystems.
-The 9600 can heat and cool 96 samples in an 8 x 12-well microplate format at a rate of approximately 1oC per second.

31. Forensic DNA – PCR ∞ Thermal Cycling Parameters: 95oC 10:00 72oC 65oC
0:15
0:30
0:40
4oC ∞
1 HOLD
32 CYCLES
2 HOLDS
Activation
Denature
Extension
Final Extension
Annealing
-Most common Thermal Cycler is the GeneAmp® PCR System 9600 from Applied Biosystems.
-The 9600 can heat and cool 96 samples in an 8 x 12-well microplate format at a rate of approximately 1oC per second.
This stage is to allow sufficient time for all DNA fragments from previous cycles to finish extension.

32. Forensic DNA – PCR ∞ Thermal Cycling Parameters: 95oC 10:00 72oC 65oC
0:15
0:30
0:40
4oC ∞
1 HOLD
32 CYCLES
2 HOLDS
Activation
Denature
Extension
Final Extension
Annealing
Final Hold
-Most common Thermal Cycler is the GeneAmp® PCR System 9600 from Applied Biosystems.
-The 9600 can heat and cool 96 samples in an 8 x 12-well microplate format at a rate of approximately 1oC per second.
This stage is to slow down all the processes and help keep the solution stable. This is like putting your sample in the refrigerator.

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

35. Four Squares
Write as much information as you can about PCR relates to DNA.
Summarize PCR:
Questions or Confusions?
Applications for use or relationship to what I know now.
-Have students copy this down on the next blank page in their notebooks. Give them 5 minutes to fill it out quietly by themselves.
-After 5 minutes, allow them to partner up and share their answers. Have students write down items they may have missed by themselves.
-Do a group share. Call on a couple of groups. Have one student say one item their partner wrote and vice versa.

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.