DNA Typing Methods RFLP- restriction fragment length polymorphism. AmpliType®PM+DQA1 DNA sequencing –Mitochondrial DNA typing. STR- short tandem repeats.

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.

Restriction Digestion

The First Human Genetic Fingerprint

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.

DQA1

DQA1

AmpliType®PM+DQA1

DQA1 PM

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.

PCR Products

• PCR products are separated on agarose gel.

PCR products can be sequenced

ABI 377 DNA Sequencing Machines

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 Electrophoresis- AB 310

capillary

Syringe with polymer solution

Autosampler tray

ABI Prism 310 Genetic Analyzer

Outlet buffer

Injection electrode

Inlet buffer

Autosampler Tray

Close-up of ABI Prism 310 Sample Loading Area

Sample Vials

Electrode

Capillary

Capillary System

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-

ABI 3100 Array Detection

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

MALDI-TOF Mass Spectrometry

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

STR Primers

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.

D8S1179 D21S11 D7S820 CSF1PO

D3S1358TH01

D13S317 D16S539 D2S1338

D19S433 D18S51TPOX

VWA

AMEL D5S818 FGA

GS500 LIZ size standard

6FAM (blue)

VIC (green)

NED (yellow)

PET (red)

LIZ (orange)

AmpFlSTR® Identifiler™