1. Dye-binding/High-resolution Thermal Denaturation PCR-what makes or breaks fragment analysis Steven F. Dobrowolski, PhD

2. Why PCR is the Most Critical Issue dsDNA binding dye indiscriminately bind dsDNA –Specific Product, Undesired Product, and Dimer Product all incorporated dye with equal affinity –All products contribute to the fluorescent signal –All products contribute to the melting profile.

3. Take Home Message Specific PCR is the only means to generate meaningful DB/HRTD data

4. Polymerase Chain Reaction Alternative Formats Capillary Tube, Strip, Plate

5. Comparing Formats Capillary High surface to volume ratio Glass, rapid heat transfer Small vapor volume Broadly adjustable thermal ramping rate Tube, Strip, Plate Low surface to volume ratio Plastic, slow heat transfer Large vapor volume Minimally adjustable, slow to moderate thermal ramping rate

6. PCR in Capillaries Low volume reactions, 5-10μl is the norm Reaction chemistry is often different than the same reaction in tube/plate. (addressed tomorrow) Cycling conditions very different than the equivalent reaction in a tube/plate Adjustable ramp speed allows fine tuning of amplification. PCR in capillaries is often more robust than possible in plate/tube

8. 32 specimens Real-time Amplification

9. DB/HRTD Melting Profiles

10. Factors that effect PCR Effective Primer Design (don’t trust software) Primer Purity (salted out, cartridge, HPLC, Gel) Primer Concentration (how much is optimal) MgCl 2 Concentration (how much is optimal) Adjuvant (KCl, glycerol, DMSO, TMSO, others) Genomic DNA (concentration, chemistry, salts) Cycling Conditions (how many, anneal temp, hold time, ramp speed) Enzyme, hot-start broadly recommended

11. Issues Specific to DB/HRTD Fragment size (bp) SensitivitySpecificity < 300100% 400 – 100096.1%99.4% Total (1248 rxns)97.8%99.7% Reed and Wittwer Clin Chem 2004

12. Situations to Avoid PCR for alternative systems used precisely the same way for DB/HRTD –PCR for sequencing need not be robust –PCR for paired probes using FRET and SimpleProbe are asymmetric –PCR for TaqMan gains specificity from the probe, thus in need not be highly specific. –PCR for RFLP need not be highly specific because the restriction pattern is the final data. –SSCP, DGGE, have their own issues G:C clamps, tailing with sequencing primers, etc

13. How to avoid looking foolish when demonstrating DB/HRTD Never tell a customer adding dye to existing PCR assays is all that is necessary Never add dye Post-PCR and expect quality profiles Always see a gel of the customers PCR product before using such to demonstrate the HR-1 Always request DNA sequence characterized samples when demonstrating HR-1 Always assay duplicates when demonstrating HR-1 Always run a gel if duplicates are inconsistent

14. Steve’s DB/HRTD Mantra It’s All About The PCR