1

Forensic Science: DNA

Prof. J. T. Spencer

2

Forensic Toxicology and Alcohol

Forensic Aspects of Arson

Serology

DNA

Forensic Entomology

Fingerprints and Firearms, Arson

Fingerprints and Firearms

3

DNA

• Deoxyribonucleic Acid

• What are nucleic Acids

• Structure of DNA

• DNA vs. RNA

• “Blue print” of life

• How it “transcribes” life

• Where is it located

• Forensic Applications

4

DNA Timeline of DNA

1868 Miescher “discovers” DNA1953 Watson and Crick report double

helix structure1977 First human gene cloned.1985 Jeffreys reports VNTR DNA

sequences1985 First report of PCR method1986 Jeffreys uses DNA to solve first

murder case (Picthfork case)1987 First conviction on DNA

evidence (Andrews case)1991 STRs first reported1998 FBI starts CODIS database2005 2.5 million DNA “finger-prints”

in FBI database

5

Nucleic Acids

• Nucleic Acids - chemical carriers of genetic information (DNA and RNA).– Consist of;

• A phosphoric Acid Molecule (H3PO4)

• A five carbon Sugar (ribose)• A nitrogen containing base

CH2

CH

OH

H

OH OH

H CC

C

H

O

HO

CH2

CH

OH

H

OH H

H CC

C

H

O

HO DEOXYRIBOSEDEOXYRIBOSE RIBOSERIBOSE

6

Nucleic Acids

CH2

CHH

OH H

H CC

C

H

O

O

N

N

N

N

NH2

P

O

O

O

-1

-1

PhosphatePhosphate

SugarSugar

BaseBase

Three “Building Blocks” linked through condensation reactions to form polymers.

Nucleotide - unit of the three building blocks (phosphate, sugar, and base)

NucleotideNucleotide

7

Nucleic Acids: Bases

N

N

N

N

NH2

N

N

N

N

O

NH2

N

N

NH2

O

N

N

H3C

O

O

N

N

O

O

AdenineAdenine GuanineGuanine CytosineCytosine

ThymineThymine(DNA Only)(DNA Only)

UracilUracil(RNA Only)(RNA Only)

8

Nucleic Acids

• Nucleic Acid Polymer (i.e., DNA)

PhosphatePhosphate

SugarSugar

BaseBase

PhosphatePhosphate

SugarSugar

PhosphatePhosphate

SugarSugar

PhosphatePhosphate

BaseBase

BaseBasePolymerPolymer

9

Nucleic Acids• Nucleic Acid Polymer (i.e., DNA)

C H2

C

H

H

O

H

H

C

C

C H

O

O

N

N

NN

NH2

P

O

O

C H2

C

H

H

O

H

H

C

C

C H

O

O

N

N

NN

NH2

P

O

O

C H2

C

H

H

O

H

H

C

C

C H

O

O

N

N

NN

NH2

P

O

O

10

Nucleic Acids• Double Helix - Hydrogen Bonds

11

Nucleic AcidsDouble Helix - Hydrogen Bonds

physics.bu.edu/~neto/ Topic6.htm

N

N

N

N

N

N

N

N

N

O

N

N

N

N

O

N

N

CH3O

O

A T

GC

H

H

H

H

H

H

H

O

H

H2C

H

O

PO O

O

O

PO O

O

O

H

H2C

H

O

PO O

O

OH CH2

O

O

PO O

O

PO O

O

OH CH2

O

PO O

O

Hydrogen Bonds

Hydrogen Bonds

H

H

12

Base Pairs

• Hydrogen Bonding

13

DNA Structure

Major Groove

Minor Groove

14

DNA Structure

15

DNA Structure

QuickTime™ and aAnimation decompressor

are needed to see this picture.

16

DNA in the Cell

chromosome

cell nucleus

Double stranded DNA molecule

Individual nucleotides

17

DNA Structure • (1) Information in the order of

nucleotides in DNA can be transcribed and translated to direct the preparation of proteins in the cell.

• (2) Every three nucleotides in DNA codes for one amino acid in the formation of a protein.

• (3) Parts of DNA coding for proteins are called genes.

• (4) DNA contains very large parts that code for nothing known.

18

DNA

• Nuclear DNA– Maternal DNA– Paternal DNA

DNA

Chromosome

19

DNA: Chromosomes

20

Human Genome Project• (1) Completed in 2003.

• (2) Determined base (nucleotide) sequence of all 30,000 human genes (the order of xx million nucleotides).

• (3) Found humans share ca. 93% of our DNA code with roundworms.

• (4) Found only ca. 1.5% of our DNA codes for compounds.

21

DNA

• Transcription:– 3 Base pairs

corresponds to 1 amino acid.

– Builds Proteins– Proteins -

enzymes, all functions for life.

22

DNA Transcription

• Transcription:

23

DNA Cellular Sources• Nuclear DNA

– Located in nucleus.– inherited 1/2 from mother and 1/2 from father.

• Mitochondrial DNA – Located in mitochondria (cellular respiration).– inherited solely from mother.

24

Where is DNA

• Cellular Matter– Organ Cells– Hair Roots– Sweat– Semen– Blood (not RBCs)

• It’s the same from every source in the body.– Usually in the cell nucleus.

Mitochondrial DNA

Nuclear DNA

25

Where DNA Samples found:Blood StainsSemen StainsChewing GumStamps & EnvelopesPlant MaterialSweaty ClothingBoneHair (sheath)Fingernail ScrapingSalivaAnimal MaterialOther

26

DNA Evidence

27

Mitochondrial DNA• Lines of evidence independently suggest

that Africa is the birthplace of humankind. (East Africa or southern Africa)

• By examining DNA in living people, the origin of our species est. between 100 000 - 150 000 years before the present.

• The use of mtDNA and the non-recombining portion of the Y chromosome have shown how females and males, respectively, have contributed to the gene pool of southern African populations.

• It is possible to reconstruct the history of mutations to a common ancestor that would have lived in prehistoric times..

28

• Forensic cases - Match or eliminate suspect with evidence.

• Paternity testing - Match or eliminate possible father(s).

• Historical investigations - Czar Nicholas and Royal family.

• Missing persons investigations - Partial remains with known samples.

• Mass disasters - Identifying remains.• Military DNA Uses - Records of personnel (DNA

“dog tags”), identifying combatants, identifying wanted terrorist suspects.

• Convicted felon DNA databases - NYS and FBI databases.

Human Identity Testing

29

DNA Analysis

• Collection of Sample• Separation and Purification of DNA• Amplification (making copies of DNA)• Cutting/Separation of Fragments• Comparison and Analysis

30

DNA “Fingerprinting”• DNA used to identify individuals• Spacing between genes is different for each

individual– Collect DNA at crime scene (both evidence and

suspect DNA)– Make more DNA using “PCR” (Polymerase Chain

Reaction to “amplify” DNA) - no longer need a big sample - can be done from 1 DNA piece.

– Cut DNA with “restriction” Enzyme (next slide)– Separate DNA fragments using “gel”– Detect DNA (25- 60 bands on gel ) 1”:1,000,000

have same pattern

31

Tandem Repeats• DNA forms genes that code for proteins.• Not all DNA codes for proteins (past useless

info separating genes).• Intergene regions contain many A,T,C,G

repeats. (why?? Unknown - maybe spacers maybe discarded genetic info - about 30% of DNA code). called Tandem Repeats.Tandem Repeats.

• Forensic DNA typing uses tandem repeats.– All humans have many tandem repeats.– Great variation per person, however, in how

many and where these repeats occur.

32

Tandem Repeats• Tandem Repeats.Tandem Repeats.

• Natural selection eliminates mutations that result in non- or less viable phenotypes. The 95% of the DNA that does not encode protein is much less subject to natural selection, and the noncoding DNA has far more sequence variation (per person).

• Variable Number Tandem repeats (VNTR)

33

Tandem Repeats• Short Tandem Repeats.Short Tandem Repeats.

ACCT ACCT ACCTACCT

ACCT ACCT ACCTACCT ACCT ACCT

34

Recombinant DNA (Cutting)• Therapeutic Proteins and other useful substances

from other organisms.– Splicing and recombining genes– Gene retrieved from one organism can be

reproduced identically (cloned) in bacteria: Insulin; “Flounder genes into a Strawberry” - flounder temperature resistance into temperature sensitive strawberries

Insulin GeneCut out gene

Bacteria

Plasmid Circular DNA Cut

Insulin

Remove DNA Plasmid

Insert Insulin DNA

35

Gene Therapy• Gene therapy replaces missing or damaged proteins by

providing the gene that will direct its manufacture. This involves stuffing the gene into a viral carrier (vector) then injecting the loaded beneficial virus into the patient. Once inside the cells, the genetic material is released and begins making the protein that should cure the disease or at least alleviate symptoms.

– Cures for several problems found in 2000;• Hemophilia• Severe combined immunodeficiency disease, or SCID• Squamous cell carcinoma (enhances tumor necrosis

factor)• using DNA to carry a substance that stimulates blood

vessel growth to damaged heart tissue.

36

DNA “Fingerprinting” RFLP• Restriction enzyme cuts at ….AAGCTT…..

• Spacing between cutting sites for enzyme is different for different individuals

37

ACCT ACCT ACCT ACCT

ACCT ACCT ACCT ACCT ACCTChromosome A

Chromosome A'

Cut Cut Cut Cut Cut

Cut Cut Cut Cut

4 kbp

4 kbp6 kbp

6 kbp

2.5 kbp

7.5 kbp

5 kbpFragments

Fragments

kbp

Chromosome A Chromosome A'

8

7

6

5

4

3

2

38

DNA “Fingerprinting”• Restriction enzyme cuts at ….AAGCTT…..

• Spacing between cutting sites for enzyme is different for different individuals

Person A Person B1 2 3 4 5 6

Cut sitesDNA DNA

12

3

456

Different DNA lengths1, 2, 3 not equal to 4, 5, 6

Separate by Gel Electrophoresis

39

40

41

DNA Separation Mechanism

+-DNA-

DNA-

DNA-DNA- DNA-

• Size based separation due to interaction of DNA molecules with entangled polymer strands

• Polymers are not cross-linked (as in slab gels)• “Gel” is not attached to the capillary wall• Polymer length and concentration determine the

separation characteristics

42

Gel Electrophoresis

(1) Amplify DNA sample, react with restriction enzymes, prepare sample

(2) Spot on Gel

(3) Use elec. Current to move DNA pieces (DNA negative, smaller pieces travel fastest, detect using radioactivity)

43

Gel ElectrophoresisLanes 1-3 contain DNA samples, perhaps from the same stock of DNA digested with 3 different Restriction Enzymes. Lanes 4 and 5 are called Standard Lanes, or sometimes Molecular Weight Markers or "Ladders". They are similar to the Control in an experiment, because we know exactly how they are going to turn out every time. These lanes contain DNA of a specific, predetermined size from a known source, digested with a Restriction Enzyme that cuts that piece of DNA a known number of times, yielding a predicted number of bands whose size we know exactly. For example,Lane 5 is DNA from the E. coli bacteriophage Lambda, digested with Hin dIII. We know exactly how long each of the visible fragments are.

44

RFLP Analysis• Enzymes break DNA into

restriction fragments • Measurements taken of

fragments that vary in length bet’n people (length polymorphism)

• Can produce extremely low random match probabilities

• Requires relatively large fresh samples (>50 ng DNA)

• Slow and expensive

45