Laboratory of Human Genetics and Reproductive Biology Brandeis University, Waltham, Massachusetts

1. LATE-PCR and Allied Technologies Principles and Applications Co-Investors Cristina Hartshorn, Ph.D. Kenneth Pierce, Ph.D. Arthur Reis, Ph.D. John Rice, M.S. Jesse Salk, B.A. J. Aquiles Sanchez, Ph.D. Lawrence J. Wangh, Ph.D. Laboratory of Human Genetics and Reproductive Biology Brandeis University, Waltham, Massachusetts Faculty Bazaar, 08/29/06 Wangh Laboratory, Brandeis University

2. RT -PCR cDNA Libraries And Expression Studies A Whole System Approach LATE-PCR PurAmp Unique Reagent to Improve PCR Reaction RNA and DNA Sample Preparation ELIXIR Samplelyse Advance Asymmetric PCR Automated Detections Systems Improved Analytical Methods Diagnostic Applications BioDefense Dilute’N Go Sequencing Infectious agent testing Genetic Testing Cancer Testing Dideoxy Sequencing Human Testing Paternity Micrometastasis Low Temperature Mis-match tolerannt Probes Sequencing by Syn. Blood Supply Testing Clinical mutations Therapy Monitoring MassArray systems Food and Consumables Testing Prenatal diagnostics Quantitative End-Point Analysis Preimplantation Genetics Forensics Wangh Laboratory, Brandeis University

3. Probe Tm Primer Tm’s Primer Annealing Probe Detection 2n molecules Symmetric PCR uses EquiMolar Primers with Similar Tm’s: Yields dsDNA Exponentially Strand Strand Denaturation Denaturation n cycles Temperature Temperature Primer Primer Extension Extension Primer Annealing Wangh Laboratory, Brandeis University

4. Probe Tm Primer Tm’s Asymmetric PCR uses non EquiMolar Primers phase I: Yields dsDNA Exponentially Strand Denaturation Temperature Primer Extension Limited Number of DS Molecules Primer Annealing Probe Detection Wangh Laboratory, Brandeis University

5. Probe Tm n cycles Primer Tm’s Asymmetric PCR uses non EquiMolar Primers phase II: Yields Single Stranded DNA Linearly Strand Denaturation Temperature Primer Extension Primer Annealing Probe Detection n molecules Wangh Laboratory, Brandeis University

6. CT Value Freeman et al., (1999) Biotechniques 26: 122-125 Limitations of Real-Time PCR • Low Sensitivity for Small Number of Initial DNA Targets Freeman et al., (1999) Biotechniques 26: 122-125 • Lack of Reliability Among Replicate Samples • No Quantitative End-Point Analysis! Wangh Laboratory, Brandeis University

7. Slope = -3.50 CT Values 3000 2500 ` Log Starting Quantity 2000 10,000 Genomes 1,000 Genomes 100 Genomes 10 Genomes Pierce et al. (2005) PNAS 102:8609-8614 Efficient Initial Amplification 1500 Fluorescence 1000 500 CT Values 0 20 30 40 50 60 70 80 Cycle Number Wangh Laboratory, Brandeis University

8. 10,000 Genomes 1,000 Genomes 100 Genomes 10 Genomes All Reactions Progress toward the Same Final Concentration of ss-DNA 3000 2500 2000 Fluorescence 1500 1000 500 CT Values 0 20 30 40 50 60 70 80 Cycle Number Wangh Laboratory, Brandeis University

9. 0.1 1 Cleanup of LATE-PCR Products by Dilution dsDNA molecules dNTP Unincorporated Excess Primer ssDNA molecules Unincorporated dNTPs Wangh Laboratory, Brandeis University

10. Amplicons Individually Pentaplex Replicates P53 611 bp -- HV1 560 bp -- TSD 452 bp -- G269 312 bp -- Globin 192 bp -- Pentaplex that Generates All Five Products Simultaneously Wangh Laboratory, Brandeis University

11. Globin Globin From Individual Amplicons From Pentaplex Amplicons G269 G269 P53 P53 TSD TSD HV1 HV1 Dilute-’N’-Go Sequencing for Product Detection following Multiplexing Wangh Laboratory, Brandeis University

12. LATE-PCR Uncouples Annealing and Detection Symmetric PCR Strand Denaturation Primer Extension Temperature Annealing And Detection Wangh Laboratory, Brandeis University

13. Low-Tm Probes High-Tm Probes LATE-PCR Uncouples Annealing and Detection LATE-PCR (linear phase) Strand Denaturation Primer Extension Temperature Annealing Detection Wangh Laboratory, Brandeis University

14. The Frequency of Sequence Differences Varies Across the Genome Forensics: Differences Among Individuals Border Control: Species Differences Medical Diagnostics: Inherited Diseases Border Control: Species Differences Health Care: Aging and Cancer, changes within a lifetime Wangh Laboratory, Brandeis University

15. Barrett’s Esophagous: Premalignant Neoplastic Growth Wild and Hardie, Nature Reviews Cancer (2003) 3:676-85 Collaboration with Brian Reid’s Laboratory Wangh Laboratory, Brandeis University

16. Early Eight Cell – Blastomeres Recovered In Pairs Wangh Laboratory, Brandeis University