DNA Typing Methods
• RFLP- restriction fragment length polymorphism.
• AmpliType®PM+DQA1
• DNA sequencing– Mitochondrial DNA typing.
• STR- short tandem repeats (PCR based).
DNA Typing and Criminal Investigations
• 1980s- Lynda Mann and Dawn Ashworth.• Richard Buckland- kitchen porter.
– Confessed to the second murder.
• Scotland Yard.– Semen samples and blood from Buckland taken.
• Dr. Alec Jeffreys at Leicester University.– DNA Fingerprinting.– Buckland was not the rapist/murderer!
• Voluntary blood submission and DNA testing.– Colin Pitchfork and Ian Kelly- bakery workers.– Kelly bragged about the blood switch.
• Colin Pitchfork was arrested and subsequently confessed in 1987.
• DNA analysis confirmed match.
Restriction Enzymes
• An enzyme that cuts DNA at specific internal sites in the nucleotide sequence.
• They recognize specific double stranded sequences in DNA.
• Recognition sites are 4-8 base pairs (bp) long.• Recognition sites are palindromes.• Arrows indicate cut sites.
Gel Electrophoresis
• Technique used to separate DNA on the basis of size (number of base pairs).
• DNA samples (mixed with loading dye) are loaded into wells.
• Samples are pushed through a gel (agarose or acrylamide) while under the influence of an electrical field.
• DNA is negatively charged due to phosphate groups and thus migrates towards the positive electrode (anode).
• The larger the DNA, the slower it travels.
PCR- polymerase chain reaction
• Kary Mullis- Nobel Prize in chemistry in 1993.
• PCR is used to target and replicate any segment of DNA.– Copy high numbers (1 trillion)
of target in 2-3 hours.• A technique that involves
repeated cycles of 3 steps:– Denature– Anneal– Extension
Make copies (extend primers)
Starting DNA Template
5’
5’
3’
3’
5’
5’
3’
3’
Add primers (anneal) 5’3’
3’5’
Forward primer
Reverse primer
DNA Amplification with the Polymerase Chain Reaction (PCR)
Separate strands
(denature)
5’
5’3’
3’
Thermal Cycling Temperatures
94 oC
60 oC
72 oC
Time
Tem
per
atur
e
Single Cycle
Typically 25-40 cycles performed during PCR
94 oC 94 oC 94 oC
60 oC60 oC
72 oC72 oC
PCR Components
• Taq DNA Polymerase- catalyzes the attachment of the nucleotides to the growing strand of DNA.
• DNTPs- deoxynucleotide triphosphates (A,T,G,C).
• MgCl2- required to activate Taq Polymerase.
• Buffer- salt and pH balanced.• Primers- a single-stranded short chain of
nucleotides (10-30 nucleotides in length).• DNA Template• Water
PCR Reaction• 25 µL reactions
– PCR tubes- thin, for rapid heat transfer.
• Thermocycler– An instrument that is programmed to repeatedly
raise and lower the temperature of a heating block.– Cycling Parameters
• 94°C for 3 minutes- initial denaturation.• 26-40 cycles of the following:
– 94 °C for 30 seconds.– 55 °C for 30 seconds.– 72 °C for 60 seconds.
• 72°C for 5 minutes- final extension.• 4°C until processing.
Inlet (cathode)
Outlet (anode)
Capillary Electrophoresis (CE)
Argon Ion Laser
Fill with Polymer Solution
Fill with Polymer Solution
50-100 m x 27 cm50-100 m x 27 cm
5-20 kV5-20 kV
- +Burn capillary window
Data Acquisition and AnalysisData Acquisition and Analysis
DNA Separation occurs in minutes...
DNA Separation occurs in minutes...
capillary
Syringe with polymer solution
Autosampler tray
ABI Prism 310 Genetic Analyzer
Outlet buffer
Injection electrode
Inlet buffer
DNA Separation Mechanism
• Size based separation due to interaction of DNA molecules with entangled polymer strands.
• Pumpable polymer can be replaced after each run.
• Polymer length and concentration determine the separation characteristics.
+-DNA-
DNA-
DNA-DNA- DNA-
Fluorescent Dyes Used in 4-Color Detection
FAM (Blue) JOE (Green)
TAMRA (Yellow) ROX (Red)NED
FL
CXR
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
Short Tandem Repeats (STRs)
• The repeat region is variable between samples while the flanking regions where primers anneal are conserved.– Homozygote- both alleles are the same length.– Heterozygote- alleles differ in length.
7 repeats
8 repeats
AATG
Multiplex PCR
• Multiple PCR reactions are run simultaneously in 1 tube.– Up to 16 reactions.– <1 ng sensitivity.
• Different fluorescent dyes used to distinguish STR alleles with overlapping size ranges.
CSF1PO
D5S818
D21S11
TH01
TPOX
D13S317
D7S820
D16S539 D18S51
D8S1179
D3S1358
FGA
VWA
13 CODIS Core STR Loci
AMEL
AMEL
Sex-typing
Position of Forensic STR Markers on Human Chromosomes
Fluorescent Labeling of STR PCR Products
• Dyes are attached to one primer in a pair used to amplify a STR marker.
• Dye-labeled oligonucleotide is incorporated into PCR product during multiplex PCR amplification giving a specific color “tag” to each PCR product.