qPCR SNAPSHOTS LIVE MAY 13, 2003

Agenda Introduction Objectives & Methodologies Detection Chemistries Instrumentation Conclusion

Real-Time PCR Ability to detect PCR product as it is synthesized Requires fluorescence-based detection chemistries and specialized detection instrumentation Advantages:  Increased analytical sensitivity  Faster results  Broad applicability Introduction

Study Demographics Introduction <1% 2% 5% 8% 18% 21% 44% 0%5%10%15%20%25%30%35%40%45% Academic Pharmaceutical/Biotechnology Hospital or University Medical Center Government Private Research Contract Research Commercial Testing Lab Medical Device/Diagnostics Healthcare Network/Facility

Quantitative vs. Qualitative Applications Quantitative only 31% Both (qualitative and quantitative) 58% Qualitative only 11% Objectives & Methodologies

Template Use DNA, cDNA and RNA as templates 34% Only RNA or cDNA as a template 49% Only DNA as a template 17% Objectives & Methodologies

Research Objectives 4% 11% 13% 10% 14% 10% 13% 14% 5% 9% 11% 10% 13% 17% 24% 0%5%10%15%20%25%30% SNP genotyping Other Allelic discrimination Gene duplication or other DNA quantification Bacterial detection/ identification Viral load detection Gene expression-confirmation of microarray data Gene expression- primary validation DNA, cDNA and RNA as a template Only DNA as a template Objectives & Methodologies

Kits vs. Components: Preferences by Template Objectives & Methodologies Individual components 34% A commercially available qRT-PCR kit 53% Both 13% Both 22% A commercially available qPCR kit (or qPCR master mix) 57% Individual components 21% DNA RNA

Kits vs. Components: Reasons for Selection Objectives & Methodologies Convenience Guaranteed optimized system Cost effective Greater flexibility Less expensive Have own system Too little reagent volume in kits KitsComponents

Detection Chemistry Preferences Detection Chemistries Both SYBR Green and fluorescent probes or primers 33% Fluorescent probes or primers only 36% SYBR Green only 31%

Commonly Used Fluorescent Probes Detection Chemistries 2% 3% 9% 15% 19% 78% 0%10%20%30%40%50%60%70%80% TaqMan probes Molecular Beacons FRET probes LUX fluorogenic primers MGB Eclipse probes Other Scorpion probes

Sensitivity Linear dynamic range Time-to-results Throughput Software Block/sample capacity Size User interface Factors Influencing Instrumentation Purchase Performance ParametersProduct Specifications

Commonly Used Real-Time PCR Instrumentation Instrumentation <1% 1% 2% 3% 7% 9% 12% 16% 18% 26% 0%5%10%15%20%25%30% ABI PRISM 7700 Sequence Detection System ABI PRISM 7000 Sequence Detection System LightCycler System iCycler iQ Real Time PCR Detection System ABI PRISM 7900HT Sequence Detection System GeneAmp 5700 Sequence Detection System Smart Cycler System DNA Engine Opticon (or Opticon 2) System Rotor-Gene Mx4000 Multiplex Quantitative PCR System Other R.A.P.I.D. System

Conclusions Real-time PCR is dominated by gene expression studies and quantitative applications. The majority of researchers prefer using kits rather than individual components for real-time PCR amplification. Fluorescent probes/primers and SYBR Green dye are nearly equal in popularity as detection chemistries. TaqMan probes are the most popular choice for users of fluorescent probes/primers. Software, sensitivity and user interface are the most important features of real-time PCR instrumentation.