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Progression of DNA Typing Markers

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.

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Progression of DNA Typing Markers

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  1. 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

  2. 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

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

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

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

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

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

  8. 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

  9. STR Analysis by Hybridization on Microchips

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

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

  12. Gels Time-of-Flight Mass Spectrometry 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

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

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