Progression of DNA Typing Markers RFLP –multilocus VNTR probes –single locus VNTR probes ( 32 P and chemiluminescence) PCR –DQ-alpha (reverse dot blot)

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Presentation transcript:

Progression of DNA Typing Markers RFLP –multilocus VNTR probes –single locus VNTR probes ( 32 P 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 10 9 individuals) RFLP requires a minimum of 25 ng of relatively undegraded DNA ( ,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

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

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

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

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

ABI 310 Result 9.3 allele: 1071 sec 10 allele: 1073 sec ABI 310 Result 9.3 allele: 1071 sec 10 allele: 1073 sec Mass Spec Result 9.3 allele:  sec 10 allele:  sec Mass Spec Result 9.3 allele:  sec 10 allele:  sec Allele 10 COfiler TM size: 187 bp MS size: 83 bp

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)

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

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