1. Forensic DNA Analysis (Part I)
Dr. Jason Linville
University of Alabama at Birmingham

2. Summary What is DNA? Where is DNA found in the body?
How does DNA differ among individuals?
Forensic DNA Analysis
DNA and Statistics

3. What is DNA?

4. What is DNA? What does DNA stand for? What does DNA do?
Deoxyribose Nucleic Acid or Deoxyribonucleic Acid
What does DNA do?
DNA contains genetic information.
DNA codes for the proteins our bodies make that are necessary for survival.

5. AGC TAG CTT ATA CTC TAT CTC TTT
What is DNA?
DNA is a code for making proteins.
AGC TAG CTT ATA CTC TAT CTC TTT
Amino
Acid
Amino
Acid
Amino
Acid
Amino
Acid
Amino
Acid
Amino
Acid
The order of amino acids determines what type of protein is made.

6. Some common proteins are:
What is DNA?
Some common proteins are:
Hemoglobin - carries oxygen from lungs to cells
Insulin - regulates metabolism
Many types of enzymes - catalyze reactions in the body, such as the breakdown of sugar for energy
DNA also determines how much of these proteins each cell makes.

7. What does DNA look like? Double Helix What is DNA?
Like a Twisted Ladder

8. What does DNA look like? What is DNA? Sugar Phosphate Backbone
(Sides of Ladder)
Nitrogenous
Base
(Rungs of Ladder)

9. The DNA ladder is made up of building blocks called nucleotides.
What is DNA?
The DNA ladder is made up of building blocks called nucleotides.
What is a nucleotide?
Adenine
Cytosine
Guanine
Thymine
Phosphate Group
Base
Deoxyribose sugar

10. What is DNA?
The 4 Bases A
Adenine C
Cytosine T
Thymine G
Guanine

11. What is DNA?
The 4 Bases A C T G

12. The 4 Bases A pairs with T G pairs with C
What is DNA?
The 4 Bases
The bases pair up to form the rungs of the ladder.
A pairs with T
G pairs with C

13. AAGTCGATCGATCATCGATCATACGT
What is DNA?
DNA is written as the sequence of these bases:
AAGTCGATCGATCATCGATCATACGT
Only one side of the ladder is written.
In humans, there are three billion (3,000,000,000) base pairs (letters) in the DNA within each cell.

14. What is DNA?
Among humans, most of the 3 billion bases in the DNA sequence are exactly the same.
Our Human DNA is 99.8% similar to each other, but the 0.2% difference is more than enough to distinguish us from one another.
Human DNA is even 98% similar to chimpanzees.
NO TWO PEOPLE HAVE IDENTICAL DNA*
*except identical twins

15. Stupid Facts: What is DNA?
If two different people started reciting their individual genetic code at a rate of one letter per second, it would take almost eight and a half minutes before they reached a difference.
If unwound and tied together, the strands of DNA in one cell would stretch almost six feet but would be only 50 trillionths of an inch wide.
If all the DNA in your body was put end to end, it would reach to the sun and back over 600 times (100 trillion times six feet divided by 92 million miles).

16. Where is DNA?

17. Where is DNA? DNA is found in the cells in our body. Nucleus
(Brain of the cell)
Mitochondria
(more later)

18. All types of cells in our body contain a copy of the same DNA.*
Where is DNA?
All types of cells in our body contain a copy of the same DNA.*
Some cells important to forensic science are:
White Blood Cell
*Sperm Cell
Cheek Cell

19. DNA in the nucleus is packaged into Chromosomes.
Where is DNA?
DNA in the nucleus is packaged into Chromosomes.

20. There are 46 chromosomes in each cell.
Where is DNA?
Chromosomes
come in pairs
(one from Mother)
(one from Father)
There are 46 chromosomes in each cell.
(23 pairs)

21. DNA can be recovered from any substance that contains cells.
Where is DNA?
What are sources of DNA at a crime scene?
DNA can be recovered from any substance that contains cells.
Blood
Semen
Saliva
Tissue
Bone
Teeth
Hair
Maggot Crops

22. Where is DNA?
Maggot Crop

23. How does DNA differ among Humans?

24. There are several ways the sequence can be different.
How does DNA differ among humans?
DNA is a sequence of 4 possible letters A G C T
Of the 3 billion letters, 99.8% of the sequence in all humans is identical.
There are several ways the sequence can be different. 

25. One of the bases (letters) can be different.
How does DNA differ among humans?
One of the bases (letters) can be different.
Person 1 AGCTAGATCGTTATTCCGAG
Person 2 AGCTAGATCGTCATTCCGAG

26. Bases (letters) can be added or removed.
How does DNA differ among humans?
Bases (letters) can be added or removed.
Person 1 AGCTAGATCGTTATTCCGAG
Person 2 AGCTAGATCGTATTCCGAG
Person 3 AGCTAGATCGTTTATTCCGAG
Person 4 AGCTCCGAG

27. Bases (letters) can be added or removed.
How does DNA differ among humans?
Bases (letters) can be added or removed.
Person 1 AGCTAGATCGTTATTCCGAG
Person 2 AGCTAGATCGTATTCCGAG
Person 3 AGCTAGATCGTTTATTCCGAG
Person 4 AGCTCCGAG

28. Regions of DNA can be repeated a different # of times.
How does DNA differ among humans?
Regions of DNA can be repeated a different # of times.
Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..

29. Regions of DNA can be repeated a different # of times.
How does DNA differ among humans?
Regions of DNA can be repeated a different # of times.
Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..
Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..

30. Forensic DNA Analysis (Part II)
Dr. Jason Linville
University of Alabama at Birmingham

31. Summary What is DNA? Where is DNA found in the body?
How does DNA differ among individuals?
Forensic DNA Analysis
DNA and Statistics

32. Forensic DNA Analysis

33. Collection of Evidence
Forensic DNA Analysis
Collection of Evidence
Types of Unknown Samples:
Blood, Semen, Stains, Saliva
Hair, Tissue, Bones, Teeth
Types of Known Samples:
Blood or buccal swabs from suspect or victim or other known person

34. Beware of Contamination
Forensic DNA Analysis
Beware of Contamination
Contamination occurs when DNA from another source gets mixed in with the sample being collected.
An investigator touches, sneezes, bleeds on a sample.
Wear gloves and use disposable instruments
Package items separately.
Especially, do not mix known samples (from victim or suspect) with unknown samples.

35. Packaging Evidence Package each item individually.
Forensic DNA Analysis
Packaging Evidence
Package each item individually.
Put evidence into paper bags, not plastic.
Moisture degrades DNA; air dry samples.
Keep samples at room temperature and out of sun.

36. Brief History of DNA - (1985)
Forensic DNA Analysis > History
Brief History of DNA - (1985)
Multilocus
RFLP
Detects VNTRs:
Variable Number of
Tandem Repeats

37. Brief History of DNA (Late 80s, Early 90s)
Forensic DNA Analysis > History
Brief History of DNA (Late 80s, Early 90s)
Single locus RFLP
D2S44 probe
Lanes 6 and 10 match
Lanes 8 and 11 match

38. Brief History of DNA (Early 90s)
Forensic DNA Analysis > History
Brief History of DNA (Early 90s)
PCR Strips (DQ alpha)
6 Alleles:
1.1, 1.2, 1.3
2, 3 or 4
A person can
have one or
two of these
numbers.

39. Two main types (90s - Present):
Forensic DNA Analysis > History
Two main types (90s - Present):
Short Tandem Repeats (STRs)
Individual identification possible
Samples: Blood stains, semen
Mitochondrial DNA
Used in cases of severely degraded DNA
Individual identification not possible
Samples: Bones, hair shafts

40. Short Tandem Repeats (STRs)
Forensic DNA Analysis > STR
Short Tandem Repeats (STRs)
Currently the most used of all forensic markers
Individual identification possible
Type of data used in the FBI CODIS database
People differ in length at these loci
Are located in the nuclear DNA (chromosomes)

41. Short Tandem Repeats (STRs)
Forensic DNA Analysis > STR
Short Tandem Repeats (STRs)
Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..
Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..

42. CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC
Forensic DNA Analysis > STR
Locus or Loci:
Refers to the location on the chromosome.
Allele:
Refers to the type of DNA.
For STRs,
the allele will be the number of repeats.
CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC

43. Example: Locus: D5S818 Alleles: 7,9 Forensic DNA Analysis > STR
Paternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATCC
Maternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC

44. Forensic DNA Analysis > STR
13 loci used in CODIS

45. Basic Steps in Analysis
Forensic DNA Analysis > STR
Basic Steps in Analysis
Extraction:
Separates DNA from sample
Amplification or PCR:
Amplifies small portions of DNA (STR regions)
Separation:
Separates amplified fragments according to size.

46. Basic Steps in Analysis
Forensic DNA Analysis > STR
Basic Steps in Analysis
Extraction:
Separates DNA from sample
Amplification or PCR:
Amplifies small portions of DNA (STR regions)
Separation:
Separates amplified fragments according to size.

47. Basic Steps in Analysis
Forensic DNA Analysis > STR
Basic Steps in Analysis
Extraction:
Separates DNA from sample
Amplification or PCR:
Amplifies small portions of DNA (STR regions)
Separation:
Separates amplified fragments according to size.

48. PCR Hood

49. The Thermal Cycler
Amplifies DNA

50. Basic Steps in Analysis
Forensic DNA Analysis > STR
Basic Steps in Analysis
Extraction:
Separates DNA from sample
Amplification or PCR:
Amplifies small portions of DNA (STR regions)
Separation:
Separates amplified fragments according to size.

51. FMBIO
Separates and Measures Amplified DNA

52. Forensic DNA Analysis > STR
Color image of gel

53. Black and white image of STR gel.
Forensic DNA Analysis > STR
Gel Electrophoresis
Black and white image of STR gel.
Samples will have one or two bands at each loci.

54. Separates and Measures Amplified DNA
ABI 310 Genetic Analyzer
Separates and Measures Amplified DNA

56. Capillary Electrophoresis
Forensic DNA Analysis > STR
Capillary Electrophoresis
Sample will have one or two peaks at each loci.

57. Compare to a ladder that has all peaks at each loci.
Forensic DNA Analysis > STR
Compare to a ladder that has all peaks at each loci.

59. DNA Profiles are compared
Forensic DNA Analysis > STR
DNA Profiles are compared
TPOX CSF1PO D5S818 D8S1179
Blood stain , , , ,8
Suspect 1 8, , , ,12
Suspect , , , ,12
Suspect 3 7, , , ,8

60. DNA Profiles are compared
Forensic DNA Analysis > STR
DNA Profiles are compared
TPOX CSF1PO D5S818 D8S1179
Blood stain , , , ,8
Suspect 1 8, , , ,12
Suspect , , , ,12
Suspect 3 7, , , ,8

61. Forensic DNA (mitochondria)
Mitochondria - The powerhouse of the cell.
Mitochondria have
their own DNA
Mitochondria

62. Mitochondrial DNA Forensic DNA Analysis > Mitochondrial Ring of DNA
YES
Double Helix
YES
Chromosomes NO

63. Mitochondrial DNA is only 16,569 letters long
Forensic DNA Analysis > Mitochondrial
Mitochondrial DNA is only 16,569 letters long
[compared to 3 billion in nuclear DNA]
There is a 900 base pair region with a 1.7% difference [D loop]

64. MtDNA used for old or degraded samples
Forensic DNA Analysis > Mitochondrial
Nuclear DNA vs. Mitochondrial DNA
Double Helix
Double Helix
46 Chromosomes
One Ring
Multiple copies in each mitochondria
One copy per cell
Multiple mitochondria in each cell
MtDNA used for old or degraded samples

65. Different colored peaks correspond to a different base
Forensic DNA Analysis > Mitochondrial
Nuclear DNA: Length is measured
mtDNA: Sequence is examined
Different colored peaks correspond to a different base

66. Basic Steps in Analysis
Forensic DNA Analysis > Mitochondrial
Basic Steps in Analysis
Extraction:
Separates DNA from sample
Amplification or PCR:
Amplifies small portions of DNA (STR regions)
Sequencing:
Sequence of letters for amplified fragments

67. AGCTAGATCGTTATTCCGAG
Forensic DNA Analysis > Mitochondrial
DNA Sequences are compared
AGCTAGATCGTTATTCCGAG
Hair Sample
AGCTAGATCGTTATTCCGAG
Victim
Conclusion: Hair may have come from the victim.

68. AGCTAGATTGTTATTCCGAG
Forensic DNA Analysis > Mitochondrial
DNA Sequences are compared
AGCTAGATTGTTATTCCGAG
Hair Sample
AGCTAGATCGTTATTCCGAG
Victim
Conclusion: Hair did not come from the victim.

69. AGCTAGATTGTTATTCCGAG
Forensic DNA Analysis > Mitochondrial
DNA Sequences are compared
AGCTAGATTGTTATTCCGAG
Cigarette
AGCTAGATCGTTATTCCGAG
Suspect #1
AGCTAGATTGTTATTCCGAG
Suspect #2
AGCTTGATTGTTATTCCGAG
Suspect #3
AGCTAGATTGTTATTCCGAG
Suspect #4
Conclusion: Cigarette could be from Suspect #2, Suspect #4 or other person with the same sequence.

70. DNA and Statistics The final result is presented as a statistic.
Do not say:
“The DNA in the bloodstain is John Doe’s DNA.”
Do say:
“The chance that another person has this DNA in the bloodstain is 1 in 300 billion.”

71. Where do the statistics come from?
Forensic DNA Analysis > Statistics
Where do the statistics come from?
First, the frequency of each allele is estimated using data from a population data base.
Allele frequency
from database
Locus: D5S818
7 26%
Alleles: 7,9
9 11%

72. Where do the statistics come from?
Forensic DNA Analysis > Statistics
Where do the statistics come from?
Next, the frequency of the genotype at each locus is calculated.
Genotype
frequency
Locus: D5S818
Alleles: 7,9
7,9 6%

73. For total frequency, multiply all of the frequencies together.
Forensic DNA Analysis > Statistics
For total frequency, multiply all of the frequencies together.
D5 = 6%
D8 = 12%
D18 = 0.5%
Total = 0.004%