Forensic DNA Analysis (Part II)

1
Forensic DNA Analysis (Part II)
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
Forensic DNA Analysis
4
Reading of Chapter 13
If material is more complicated than what we
cover in class, it likely wont be on a quiz or
exam.
5
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

6
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.

7
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.

8
Forensic DNA Analysis gt History

• Brief History of DNA - (1985)

Multilocus RFLP
Detects VNTRs Variable Number of Tandem Repeats
9
Forensic DNA Analysis gt History

• Brief History of DNA (Late 80s, Early 90s)

Single locus RFLP D2S44 probe Lanes 6 and 10
match Lanes 8 and 11 match
10
Forensic DNA Analysis gt 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.
11
Forensic DNA Analysis gt 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

12
Forensic DNA Analysis gt 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)

13
Forensic DNA Analysis gt STR

• Short Tandem Repeats (STRs)

Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5 6
Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..
1 2 3 4 5
Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..
1 2 3 4 5 6 7
14
Forensic DNA Analysis gt 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
15
Forensic DNA Analysis gt STR
Example
Locus D5S818
Alleles 7,9
Paternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATCC
Maternal chromosome 5
CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC
16
Forensic DNA Analysis gt STR
13 loci used in CODIS
17
Forensic DNA Analysis gt 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.

18
Forensic DNA Analysis gt 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.

19
Forensic DNA Analysis gt 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.

20
PCR Hood
21
The Thermal Cycler
Amplifies DNA
22
Forensic DNA Analysis gt 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.

23
FMBIO
Separates and Measures Amplified DNA
24
Forensic DNA Analysis gt STR

• Color image of gel

25
Forensic DNA Analysis gt STR
Gel Electrophoresis
Black and white image of STR gel.
Samples will have one or two bands at each loci.
26
ABI 310 Genetic Analyzer
Separates and Measures Amplified DNA
27
(No Transcript)
28
Forensic DNA Analysis gt STR
Capillary Electrophoresis

• Sample will have one or two peaks at each loci.

29
Forensic DNA Analysis gt STR

• Compare to a ladder that has all peaks at each
  loci.

30
(No Transcript)
31
Forensic DNA Analysis gt STR
DNA Profiles are compared
TPOX CSF1PO D5S818 D8S1179
Blood stain 7,9 10,13 7,15
8,8
Suspect 1 8,9 10,10 9,10 11,12
Suspect 2 10,11 9,13 8,14
9,12
Suspect 3 7,9 10,13 7,15 8,8
32
Forensic DNA Analysis gt STR
DNA Profiles are compared
TPOX CSF1PO D5S818 D8S1179
Blood stain 7,9 10,13 7,15
8,8
Suspect 1 8,9 10,10 9,10 11,12
Suspect 2 10,11 9,13 8,14
9,12
Suspect 3 7,9 10,13 7,15 8,8
33
Forensic DNA (mitochondria)

• Mitochondria - The powerhouse of the cell.

Mitochondria have their own DNA
Mitochondria
34
Forensic DNA Analysis gt Mitochondrial

• Mitochondrial DNA

Ring of DNA YES
Double Helix YES
Chromosomes NO
35
Forensic DNA Analysis gt 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
36
Forensic DNA Analysis gt Mitochondrial

• Nuclear DNA vs. Mitochondrial DNA

Double Helix
Double Helix
One Ring
46 Chromosomes
Multiple copies in each mitochondria
One copy per cell
Multiple mitochondria in each cell
MtDNA used for old or degraded samples
37
Forensic DNA Analysis gt Mitochondrial
Nuclear DNA Length is measured mtDNA
Sequence is examined
Different colored peaks correspond to a different
base
38
Forensic DNA Analysis gt 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

39
Forensic DNA Analysis gt Mitochondrial

• DNA Sequences are compared

AGCTAGATCGTTATTCCGAG
Hair Sample
AGCTAGATCGTTATTCCGAG
Victim
Conclusion Hair may have come from the victim.
40
Forensic DNA Analysis gt Mitochondrial

• DNA Sequences are compared

AGCTAGATTGTTATTCCGAG
Hair Sample
AGCTAGATCGTTATTCCGAG
Victim
Conclusion Hair did not come from the victim.
41
Forensic DNA Analysis gt 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.
42
DNA and Statistics

• The final result is presented as a statistic.

Do not say The DNA in the bloodstain is John
Does DNA.
Do say The chance that another person has this
DNA in the bloodstain is 1 in 300 billion.
43
Forensic DNA Analysis gt 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
44
Forensic DNA Analysis gt 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
45
Forensic DNA Analysis gt Statistics

• For total frequency, multiply all of the
  frequencies together.

D5 6 D8 12 D18 0.5 Total 0.004
46
(No Transcript)
47
(No Transcript)
48
Demonstration

• Calculating Frequencies