Dna forensic

Forensic DNA Analysis

Summary

What is DNA?

Where is DNA found in the body?

How does DNA differ among individuals?


DNA and Statistics

What is DNA?

What is DNA?

What does DNA stand for?

What does DNA do?• DNA contains genetic information.• DNA codes for the proteins our bodies

make that are necessary for survival.

Deoxyribose Nucleic Acid or Deoxyribonucleic Acid

What is DNA?

DNA is a code for making proteins

AGC TAG CTT ATA CTC TAT CTC TTT

AminoAcid

AminoAcid

AminoAcid

AminoAcid

AminoAcid

AminoAcid

The order of amino acids determines what type of protein is made.

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.

What is DNA?

What does DNA look like?

Double Helix Like a Twisted Ladder

What is DNA?

What does DNA look like?Sugar Phosphate

Backbone(Sides of Ladder)

NitrogenousBase

(Rungs of Ladder)

What is DNA?The DNA ladder is made up of building blocks called nucleotides.

What is a nucleotide?

Phosphate Group

Deoxyribose sugar

Base

AdenineCytosineGuanineThymine

The 4 Bases

AAdenine

GGuanine

CCytosine

TThymine

The 4 Bases

G

C

T

A

The 4 Bases

A pairs with T

G pairs with C

The bases pair up to form the rungs of the ladder.

What is DNA?

DNA is written as the sequence of these bases:

AAGTCGATCGATCATCGATCATACGT

• In humans, there are three billion (3,000,000,000) base pairs (letters) in the DNA within each cell.

• Only one side of the ladder is written.

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

What is DNA?

Stupid Facts:• 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).

Where is DNA?

Where is DNA?

DNA is found in the cells in our body.

Nucleus(Brain of the cell)

Mitochondria(more later)

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

Where is DNA?

DNA in the nucleus is packaged into Chromosomes

Where is DNA?

(one from Mother)

(one from Father)

Chromosomes

come in pairs

There are 46 chromosomes in

each cell. (23 pairs)

Where is DNA?

What are sources of DNA at a crime scene?

• Blood• Semen• Saliva• Tissue

• Bone• Teeth• Hair• Maggot Crops

DNA can be recovered from any substance that contains cells.

Maggot Crop

How does DNA differ among Humans?

How does DNA differ among humans?

DNA is a sequence of 4 possible letters

GA 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.


1. One of the bases (letters) can be different.

Person 2 AGCTAGATCGTCATTCCGAGPerson 1 AGCTAGATCGTTATTCCGAG


2. Bases (letters) can be added or removed.

Person 1 AGCTAGATCGTTATTCCGAGPerson 2 AGCTAGATCGTATTCCGAGPerson 3 AGCTAGATCGTTTATTCCGAGPerson 4 AGCTCCGAG


2. Bases (letters) can be added or removed.

Person 1 AGCTAGATCGTTATTCCGAGPerson 2 AGCTAGATCGTATTCCGAGPerson 3 AGCTAGATCGTTTATTCCGAGPerson 4 AGCTCCGAG


Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..

3. Regions of DNA can be repeated a different # of times


3. Regions of DNA can be repeated a different # of times

Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..1 2 3 4 5 6

Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..

Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..

1 2 3 4 5

1 2 3 4 5 6 7



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


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.


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.


Brief History of DNA - (1985)

Multilocus

RFLP

Detects VNTRs:Variable Number of

Tandem Repeats


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

Single locus RFLP

D2S44 probe

Lanes 6 and 10 match

Lanes 8 and 11 match


Brief History of DNA (Early 90s)

PCR Strips (DQ alpha)

6 Alleles:1.1, 1.2, 1.32, 3 or 4

A person canhave one ortwo of these

numbers.


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

Two main types of analyses (90s - Present) :


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)



Regions of DNA can be repeated a different # of times

Person 1 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTTTCAT..

Person 2 ..GCCAGCTAGCTAGCTAGCTAGCTTTCAT..

Person 3 ..GCCAGCTAGCTAGCTAGCTAGCTAGCTAGCTT..

1 2 3 4 5 6

1 2 3 4 5

1 2 3 4 5 6 7



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


Locus: D5S818

Alleles: 7,9

CCAGATAGATAGATAGATAGATAGATAGATCC

Paternal chromosome 5

Maternal chromosome 5

CCAGATAGATAGATAGATAGATAGATAGATAGATAGATCC

Example


STR Marker Chromosome Repeat Sequence Repeat units Other AllelesTPOX 2 AATG 6 - 14

CSF1PO 5 AGAT 6 - 15 10.3D5S818 5 AGAT 7 -15D7S820 7 GATA 6 - 14

D8S1179 8 TATC 8 - 19D13S317 13 TATC 7 - 15D16S539 16 GATA 5, 8 - 15

D3S1358 3 TCTA* 9, 11- 20 15.2, 16.2FGA 4 CTTT* 15 - 30 16.2 -30.2

22.3, 34.2, 46.2TH01 11 AATG* 3, 5 - 12 8.3, 9.3, 10.3, 13.3VWA 12 TCTA* 11 - 22 15.2

D18S51 18 AGAA* 8 - 27 13.2, 14.2, 15.217.2, 19.2

D21S11 21 TCTA* 24 - 38 24.2 - 35.2

13 loci used in CODIS


Basic Steps in Analysis

Extraction:• Separates DNA from sample

Separation:• Separates amplified fragments according to size.

Amplification or PCR:• Amplifies small portions of DNA (STR regions)

PCR Hood






The Thermal Cycler

Amplifies DNA






FMBio

Separates Amplified DNA


Color image of gel


Black and white image of STR

gel.Samples will have one or two bands

at each loci.

Gel Electrophoresis

ABI 310 Genetic Analyzer

Separates Amplified DNA


Sample will have one or two peaks at each loci.

Capillary Electrophoresis


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



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

TPOX CSF1PO D5S818 D8S1179

Forensic DNA (mitochondria)

Mitochondria - The powerhouse of the cell.

Mitochondria

Mitochondria have their own DNA


Mitochondrial DNA

Double HelixYES

ChromosomesNO

Ring of DNAYES


Mitochondrial DNA

Mitochondrial DNA is only 16,569 letters long.

There is a 900 base pair region with a 1.7% difference (D loop).

(compared to 3 billion in nuclear DNA)


Nuclear DNA vs. Mitochondrial DNA

Double Helix Double Helix

One copy per cell Multiple copies in

each mitochondriaMultiple mitochondria in

each cell

One Ring 46 Chromosomes

MtDNA used for old or degraded samples

Forensic DNA (Mitochondria)

For nuclear DNA: Length is measured

For mtDNA: Sequence is examined

Different colored peaks correspond to a different base




Sequencing:• Sequence is determined by another reaction

and separation of sequenced fragments

Amplification or PCR:• Amplifies small portion of mtDNA (D loop)


DNA Sequences are compared to each other.

AGCTAGATCGTTATTCCGAG


Hair found onSuspect

Victim

Conclusion: Hair may have come from the victim.


DNA Sequences are compared to each other.

AGCTAGATTGTTATTCCGAG


Hair found onSuspect

Victim

Conclusion: Hair did not come from the victim


AGCTAGATTGTTATTCCGAG


Cigarette butt at crime scene

Suspect #1

Conclusion: Cigarette could be from Suspects #2, #4 or other person with the same sequence.

AGCTAGATTGTTATTCCGAGSuspect #2

AGCTTGATTGTTATTCCGAGSuspect #3

AGCTAGATTGTTATTCCGAGSuspect #4

DNA and Statistics

The final result is presented as a statistic.

Do Say:“The chance that another person has this DNA

in the bloodstain is 1 in 300 billion.”

Do not say:“The DNA in the bloodstain is John Doe’s DNA.”

DNA and Statistics

Where do the statistics come from?

First, the frequency of each allele is estimated using data from a population data base.

Locus: D5S818

Alleles: 7,9

Allele frequencyfrom database

7 26%

9 11%

DNA and Statistics

Where do the statistics come from?

Next, the frequency of the genotype at each locus is calculated.

Locus: D5S818

Alleles: 7,9 7,9 6%

Genotype frequency

DNA and Statistics

For total frequency, multiply all of the frequencies together.

D5 = 6%

D8 = 12%

D18 = 0.5%

Total = 0.004%

7 0.00 6 0.008 0.00 7 0.009 0.03 8 0.53

10 0.25 9 0.0911 0.31 10 0.0612 0.33 11 0.2813 0.06 12 0.0414 0.01 13 0.0015 0.00

CSF1PO TPOX

5 0.01 13 0.006 0.24 14 0.137 0.15 15 0.088 0.12 16 0.219 0.16 17 0.27

9.3 0.33 18 0.2010 0.01 19 0.0911 0.00 20 0.02

21 0.00

TH01 vWA

7 0.00 6 0.008 0.03 7 0.019 0.11 8 0.16

10 0.08 9 0.1511 0.32 10 0.3012 0.27 11 0.2013 0.17 12 0.1214 0.03 13 0.0615 0.00 14 0.01

D16S539 D7820

7 0.00 7 0.008 0.14 8 0.009 0.05 9 0.01

10 0.05 10 0.0611 0.31 11 0.3712 0.31 12 0.3513 0.08 13 0.1914 0.06 14 0.0115 0.00 15 0.00

D13S317 D5S818

Demonstration

Calculating Frequencies

Technology

Dna forensic