Maximizing the Efficacy of Melt Profiling through Stringent PCR Optimization

1. Maximizing the Efficacy of Melt Profiling through Stringent PCR Optimization Steven F. Dobrowolski, PhD

2. Adapting a Customer’s PCR Assay to DB/HRTDOptimizing PCR to a Capillary System and Initial Profiling Steven F. Dobrowolski, PhD

3. First QuestionIs the customer currently using the LightCycler? If yes, the job of adapting a PCR assay for melting analysis is much simpler.

4. Real-time PCR is Extremely Useful to Assess Amplification

5. Adapting a LightCycler Assay • Reaction volume of 10μl • Hot-start polymerase, Klen Taq in a complex with TaqStart antibody • Add LCGreen to PCR mastermix (1μl/reaction) and omit an equal amount of water. • Single fluorescence acquisition in Channel 1 at end of extension phase. • Use their established amplification parameters but add initial denaturation and re-annealing at end

6. Customer PCR FRET Assay 10X buffer 12 10X dNTP 12 Fwd Primer 6 Rev Primer 6 Probe 1 6 Probe 2 6 Enzyme 2.4 H20 57.6 Add 9ul + 1ul DNA PCR with LCGreen 10X buffer 12 10X dNTP 12 10X LCGreen 12 Fwd Primer 6* Rev Primer 6* Taq/antibody 12 H20 48 Add 9ul + 1 DNA Modifying a Typical PCR Cocktail10 reactions, 10μl volumes

7. Potentially SuccessfulTry Something New

8. Customer’s Assay’s are Developed for Block-Style Cycler • This is harder, but by no means impossible • Ask for a PCR assay that is robust, free of undesired product, and between 100-250 bp • Ask to see a gel of the chosen product. • Avoid a fragment that requires DMSO, Glycerol or other adjuvant in the PCR. • Chemistry and Cycling will require modifications

9. Chemistry Optimization for PCR in Capillaries- where to begin • Use a 10μl reaction volume • Use Idaho Technology buffers, start with 20mM MgCl2 • Use hot-start with Klen taq in a complex with TaqStart antibody • Use recommended primer concentration • Use 15-30ng template DNA

10. Cycling ConditionsBlock to CapillaryMajor changes required for Adaptation to PCR in a Capillary System • Cycling Protocol has 3 Components • Initial Denaturation (high temperature step) 1-2 minutes at 94-960C • Amplification protocol-where the issues are found • Re-annealing: 940C 10-20 seconds> maximum ramp to 400C, hold for 1 minute

11. The Amplification Protocol2 out of 3 are pretty easy • Denaturation: 0-2 seconds at 940C is widely applicable, at least 95% of all fragments 300 bp or less • Annealing Temperature & Hold Duration- the biggest issue in adapting a customer’s assay • Extension: 2-5 seconds at 720C is widely applicable, at least 95% of all fragments 300 bp or less

12. Anneal Temperature GradientCalculated Tm = 620C 620C 740C

13. Beginning of Melt Inflection ~900C End of Melt Inflection at ~92.10C Tm ~91.30C Difference Tm to release 0.80C Tm of primer on previous slide was not 620C Primers bind DNA and Melt as do any other dsDNA molecule

14. The Point to All of This? • Do not be intimidated by Tm estimates provided by software or oligonucleotide manufacturers • The actual Tm of a customer’s primer set is probably higher than believed • Annealing Temperature is a powerful means by which to increase specificity of PCR • Makes your melting data better.

15. Primer Concentration: a little can change a lot

16. MgCl2: it’s Influence on Product Yield and Specificity • If undesired product is present on gel reducing the MgCl2 concentration can increase specificity • If product yield is not robust, increasing MgCl2 can increase product yield

17. Increasing product yield by altering MgCl2 concentration

18. MgCl2 and its influence on melt profiling 3 mM 2 mM

19. Select 2mM Select 3mM

20. LightCycler or Block-Cycler Assay:Determining a melt window for a new fragment, ramp 0.30/sec

21. Note • 18 of last 20 slides addressed PCR • 1 slide addressed melting • Which issues is more important to a successful adaptation of a customer’s assay to DB/HRTD chemistry?

22. It’s all about the PCR