Progression of DNA Typing Markers

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Presentation transcript:

Progression of DNA Typing Markers RFLP multilocus VNTR probes single locus VNTR probes (32P and chemiluminescence) PCR DQ-alpha (reverse dot blot) PolyMarker (6 plex PCR; dots for SNPs) D1S80 (AMP-FLPs) singleplex STRs with silver staining multiplex STRs with fluorescent dyes

Changing Technologies Paradigm Shift: Restriction Fragment-Length Polymorphisms to Short Tandem Repeats Five RFLP probes provide almost exclusive identity (~ 1 in 109 individuals) RFLP requires a minimum of 25 ng of relatively undegraded DNA (1000 - 20,000 basepairs) Short Tandem Repeats (STRs) only require ~ 1 ng DNA that can be partially degraded Discrimination power: 5 RFLP probes equals ~ 12 STR loci

potential perpetrators Which Suspect, A or B, cannot be excluded from potential perpetrators of this assault?

DNA Separation occurs in minutes... Capillary Electrophoresis (CE) Fill with Polymer Solution Argon Ion Laser 50-100 m x 27 cm 5-20 kV - + Burn capillary window Inlet (cathode) Outlet (anode) DNA Separation occurs in minutes... Data Acquisition and Analysis

Capillary electrophoresis with multi-color detection capabilities ABI PRISM® 310 Genetic Analyzer Automated gel pouring Automated sample injection Capillary electrophoresis with multi-color detection capabilities

ABI Prism 310 Genetic Analyzer capillary Syringe with polymer solution Injection electrode Autosampler tray Outlet buffer Inlet buffer

Close-up of ABI Prism 310 Sample Loading Area Electrode Capillary Sample Vials Autosampler Tray

Allele 10 COfilerTM size: 187 bp MS size: 83 bp ABI 310 Result 9.3 allele: 1071 sec 10 allele: 1073 sec Mass Spec Result 9.3 allele: 203.3 sec 10 allele: 204.8 sec Allele 10 COfilerTM size: 187 bp MS size: 83 bp

Steps in DNA Analysis Collection Extraction Quantitation Genotyping DNA Quantitation Slot Blot 1 ng 0.3 ng 0.7 ng 0.5 ng No DNA Sample Collection & Storage Buccal swab Blood Stain DNA Extraction Collection Extraction Quantitation Genotyping Interpretation of Results Database Storage & Searching Specimen Storage Multiplex PCR Amplification STR Typing DNA Database Male: 13,14-15,16-12,13-10,13-15,16 Interpretation of Results

STR Analysis by Hybridization on Microchips

MWG Biotech RoboAmp 4200 Automated PCR Setup and Mass Spec Sample Preparation

Mass Spec Sample Plates PerSeptive Biosystems (100 positions) Bruker (384 positions)

DNA separations occur in microseconds! Time-of-Flight Mass Spectrometry Gels Detector DNA separations occur in microseconds! DNA Reaction Products (Size separated and drifting to the detector) Drift Region Electric-Field Free Pulsed Laser Beam High-Density Sample Array Ion Extractor Acceleration Region X-Y sample control

Bruker BIFLEX III Time-of-Flight Mass Spectrometer Capable of fully automated data acquisition on 384 or more samples per plate