Chapter 5Biochemical Forensic

Analysis: DNAProf. J. T. Spencer

Adjunct Prof. T. L. Meeks

Learning Goals and Objectives

The use of DNA evidence has become the "gold standard" of forensic investigations. In order to understand the wealth of information that can be gained from forensic DNA studies…

Learning Goals and Objectives

• The chemical structure of DNA and how it holds genetic information

• The transcription and translation processes of DNA

• What parts of DNA are involved in forensic examinations

• What are variable number tandem repeats (VNTR) and short tandem repeats (STR)

• What a mutation is• How the Restriction Fragment Length

Polymorphism (RFLP) method works

Learning Goals and Objectives

• How the polymerase chain reaction (PCR)/STR method of DNA typing works

• How frequency of occurrences of STRs in populations is determined and used

• What is CODIS• How mitochondrial DNA can be used in

forensic investigations• How DNA typing is being used in plants and

other living organisms

The Cell

• The smallest unit of life• The nucleus is the

“brain” of the cell– contains all the genetic

info the cell needs to exist & to reproduce

• In most types of cells, genetic information is organized into structures called chromosomes

Chromosomes

• In most types of cells, genetic information is organized into structures called chromosomes– usually X shaped

• Y chromosome in males

– 23 pairs in humans• one from mother & one

from father

Genes

• Each chromosome contains hundreds to thousands information blocks called genes

• Each gene is the blueprint for a specific type of protein in the body– only identical twins will have all

the genes identical

Chromosomes

• Each chromosome is a single polymeric molecule called DNA– if fully extended the

molecule would be about 1.7 meters long

– unwrapping all the DNA in all your cells

• cover the distance from earth to moon 6,000 times

Structure of DNA

Nucleotides

• DNA is a polymer built from monomers called nucleotides

• Each nucleotide is consists of– deoxyribose

• pentose sugar

– phosphoric acid– a nitrogenous base

The DNA Backbone

• The monomers are linked together by phosphodiester bridges (bonds)– links the 3’ carbon in

the ribose of one nucleotide to the 5’ carbon in the ribose of the adjacent nucleotide

The DNA Double Helix

• DNA is normally a double stranded macromolecule

• Two polynucleotide chains are held together by H-bonding– A always pairs with T– C always pairs with G

5’ T-T-G-A-C-T-A-T-C-C-A-G-A-T-C 3’

3’ A-A-C-T-G-A-T-A-G-G-T-C-T-A-G 5’

In a double helix the strands go in opposite directions

Functions of DNA

• Two Functions– To transmit information from one generation

of cells to the next – To provide the information for the synthesis of

components (proteins) necessary for cellular function

Cell Types

Where can DNA be found?Where can DNA be found?

Cell Blood

Sweat

Hair Roots Saliva

Various Tissue Semen

SAME

Nuclear DNA

Where are the types of DNA found in a cell?Where are the types of DNA found in a cell?

Mitochondrial DNACellCell

Sources of Biological Evidence

• Blood• Semen• Saliva• Urine• Hair• Teeth• Bone• Tissue

Types of objects where DNA may be foundTypes of objects where DNA may be found

Blood Stains

Semen Stains

Chewing Gum

Stamps & Envelopes

Penile Swabs

Plant Material

Sweaty Clothing

Bone

Hair

Fingernail Scraping

Saliva

Animal Material

Where DNA Evidence is Found

BloodHair RootsSalivaSweatTissue

Chemical

DNA

Isolation of DNAIsolation of DNA

Semen stainChemical

RemoveEpithelial

DNA

Differential Isolation of DNADifferential Isolation of DNA

Different Chemical

Sperm DNA

Semen stain

Epithelial DNA

Sperm DNA

DNA

Solution

AmplificationAmplification(making copies)(making copies)

TG C

ATTA

G C

A T

A

G

TA

GA

A T

C

AT

CT

Heat

Step one of a single cycle

DENATUREDENATURE

TAC TA TTCTT AT C

AA TA G G

Step two of a single cycle

ANNEALANNEAL

Step three of a single cycle

TAAATA G G

T ACTT AT C TAC TT

CA

AA

G

GG

GT

TT

T

A G

EXTENDEXTEND

1 Cycle2 Cycles

3 Cycles

4 Cycles

5 Cycles

28 Cycles

AmplificationAmplification

DNA

PCR (Polymerase Chain Reaction)PCR (Polymerase Chain Reaction)

Analysis of amplified DNA Analysis of amplified DNA

AmplifiedDNA

DNAProfile

Brief History of Forensic DNA Typing

• 1980 - Ray White describes first polymorphic RFLP marker

• 1985 - Alec Jeffreys discovers multilocus VNTR probes

• 1985 - first paper on PCR• 1988 - FBI starts DNA casework• 1991 - first STR paper• 1995 - FSS starts UK DNA database• 1996 – First mtDNA case• 1998 - FBI launches CoDIS database

DNA Use in Forensic Cases

• Most are rape cases or murders

• Looking for match between evidence and suspect

• Must compare victim’s DNA profile

•Mixtures must be resolved

•DNA is often degraded

•Inhibitors to PCR are often present

Challenges

Human Identity Testing

• Forensic cases -- matching suspect with evidence

• Paternity testing -- identifying father

• Historical investigations-Czar Nicholas, Jesse James

• Missing persons investigations• Mass disasters -- putting pieces back together

• Military DNA “dog tag”• Convicted felon DNA databases

Sample Obtained from Crime Scene or

Paternity Investigation Biology

DNAExtraction

DNAExtraction

DNAQuantitation

DNAQuantitation

PCR Amplificationof Multiple STR

markers

PCR Amplificationof Multiple STR

markers

TechnologySeparation and Detection of

PCR Products(STR Alleles)

Sample Genotype

Determination

GeneticsComparison of Sample

Genotype to Other Sample Results

Comparison of Sample Genotype to Other

Sample Results

If match occurs, comparison of DNA profile to population databases

If match occurs, comparison of DNA profile to population databases

Generation of Case Report with Probability

of Random Match

Generation of Case Report with Probability

of Random Match

Steps in DNA Sample Processing

BloodHair RootsSalivaSweatTissue

Chemical

DNA

Extraction of DNAExtraction of DNA

RFLP Analysis• Enzymes break DNA into

restriction fragments • Measurements taken of

fragments that vary in length across people (length polymorphism) because they contain VNTRs

• can produce extremely low random match probabilities

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

• slow and expensive

Which Suspect, A or B, cannot be excluded from

the class of potential

perpetrators of this assault?

DNA Fingerprinting

• The basic structure of everyone’s DNA is the same– the difference between people is the ordering of the

base pairs

• Every person can be distinguished by the sequence of their base pairs– millions of base pairs make this impractical– a shorter method uses repeating patterns that are

present in DNA

VNTR’s

• DNA strands contain information which directs an organism’s development– exons

• Also contain stretches which appear to provide no relevant genetic information– introns– repeated sequences of base pairs

• Variable Number Tandem Repeats (VNTRs)• can contain anywhere from 20 to 200 base pairs

VNTRs

• All humans have some VNTRs

• VNTRs come from the genetic information donated by parents– can have VNTRs from mother, father or a

combination

D1 = biological daughter of both parentsD2 = child of mother & former husbandS1 = couple’s biological sonS2 = adopted son

VNTR Analysis

• Usually an individual will inherit a different variant of the repeated sequence from each parent

VNTR Analysis

• PCR primers bracket the locus

• PCR reaction forms a nucleotide chain from the template

VNTR Analysis

• The length of the amplified DNA & its position after electrophoresis will depend on the number or repeated bases in the sequence

Analysis used 3 different VNTR loci for each suspect giving6 bands

Although some individuals have several bands in common, the overall pattern is distinctive for each

Suspects A & C can be eliminatedB remains a suspect

Short Tandem Repeats (STRs)

the repeat region is variable between samples while the flanking regions where PCR primers bind are constant

7 repeats

8 repeats

AATG

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another

Short Tandem Repeat

AGAT AGATAGAT

AGAT

AGAT

AGAT

AGAT

AGAT

AGAT

AGAT

6

4

DNA Profile =4,6

TCTA TCTA TCTA

TCTA

TCTA

TCTA

TCTA

TCTA

TCTA

TCTA

7

5

DNA Profile =5,7

TCTA

TCTA

STRSTR

An Example Forensic STR Multiplex Kit

D3 FGAvWA 5-FAM (blue)

D13D5 D7 NED (yellow)

A D8 D21 D18 JOE (green)

GS500-internal lane standard

ROX (red)

AmpFlSTR® Profiler Plus™Kit available from PE Biosystems (Foster City, CA)

9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction

100 bp 400 bp300 bp200 bpSize Separation

Col

or S

epar

atio

n

Short Tandem Repeats (STRs)

the repeat region is variable between samples while the flanking regions where PCR primers bind are constant

AATG

7 repeats

8 repeats

AATG AATG

Primer positions define PCR product size

Fluorescent dye label

13 CODIS Core STR Loci with Chromosomal Positions

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

AMEL

AMEL

DNA Quantitation using Slot Blot

AMEL

D3

TH01 TPOX

Penta D

Penta EFGAD21 D18

CSF

D16D7

D13D5VWA D8

PCR Amplification with Fluorescent STR Kits and Separation with Capillary Electrophoresis

Blood Stain

Overview of Steps Involved in DNA Typing

Genotyping by Comparison to Allelic Ladder

Genotyping by Comparison to Allelic Ladder

ABI Prism 310 Genetic Analyzer

capillary

Syringe with polymer solution

Autosampler tray

Outlet buffer

Injection electrode

Inlet buffer

Chemistry Involved• Injection

– electrokinetic injection process– importance of sample preparation

(formamide)

• Separation– capillary– POP-4 polymer– buffer

• Detection– fluorescent dyes with excitation and emission

traits – virtual filters (hardware/software issues)

Separation Issues

• Run temperature -- 60 oC helps reduce secondary structure on DNA and improves precision

• Electrophoresis buffer -- urea in running buffer helps keep DNA strands denatured

• Capillary wall coating -- dynamic coating with polymer

• Polymer solution -- POP-4

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• Pumpable -- can be replaced after each run• Polymer length and concentration determine the separation

characteristics

ABI 310 Filter Set FABI 310 Filter Set F

520 540 560 580 600 620 640WAVELENGTH (nm)

100

80

60

40

20

0

5-FAM JOE NED ROX

Laser excitation(488, 514.5 nm)Laser excitation(488, 514.5 nm)

No

rmal

ized

Flu

ore

sce

nt

Inte

ns

ity

Fluorescent Emission Spectra for ABI Dyes

Sample Detection

CCD Panel

ColorSeparation

Ar+ LASER (488 nm)

Fluorescence ABI Prism spectrograph

Capillary or Gel Lane

Size Separation

Labeled DNA fragments (PCR products)

Detection region

Principles of Sample Separation and Detection

Mitochondrial DNA

What is mtDNA What is mtDNA Typing?Typing?

Database and Database and statistical issuesstatistical issues

A Mitochondrial Exclusion

A Mitochondrial Inclusion

Mitochondrial Inconclusive?

The Future of Forensic DNA

CoDIS

SNP’s & Chips

FBI’s CoDIS DNA Database

Combined DNA Index System• Used for linking serial crimes and

unsolved cases with repeat offenders• Launched October 1998• Links all 50 states• Requires >4 RFLP markers

and/or 13 core STR markers• Current backlog of >600,000 samples

On August 25, 1979, an 8-year old girl was brutally raped and murdered inSan Pablo, CA. Semen was collected from the body and placed in anevidence room, where it sat for 22 years. Through this program, a DNA profilewas made and submitted to the state and federal databases. This resulted ina “cold hit” identifying Joseph Cordova Jr. as the suspect. Cordova was ahabitual child molester who at the time of the DNA analysis was incarceratedin a Colorado prison. Cordova was subsequently charged with molesting,raping and murdering the 8-year old girl.

On November 8, 2000, a 12 year old girl, was kidnapped off of the street inRancho Cordova, CA, and driven to Feather River in Sutter County where shewas sexually assaulted and then killed. Nine months later, Justin Weinbergerwas stopped for a traffic violation in New Mexico. A check by police revealedthat Weinberger was wanted on a federal warrant for child pornography. Hewas detained and voluntarily provided a DNA sample. Analysis of that DNAsample resulted in a match with evidence identifying Weinberger as the suspect in this case. Weinberger was subsequently extradited to California where he was tried and convicted of the murder of the 12-year old girl.

Cold Hits and Solved Cases

STR Analysis by Hybridization on Microchips