Chapter 5 Biochemical Forensic Analysis: DNA Prof. J. T. Spencer Adjunct Prof. T. L. Meeks
Jan 12, 2016
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