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

2. Adapting a Customers PCR Assay to DB/HRTD Optimizing PCR to a Capillary System and Initial Profiling Steven F. Dobrowolski, PhD

3. First Question Is 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. Modifying a Typical PCR Cocktail 10 reactions, 10μl volumes Customer PCR FRET Assay –10X buffer12 –10X dNTP12 –Fwd Primer 6 –Rev Primer 6 –Probe 1 6 –Probe 2 6 –Enzyme 2.4 –H 2 057.6 Add 9ul + 1ul DNA PCR with LCGreen –10X buffer12 –10X dNTP12 –10X LCGreen12 –Fwd Primer6* –Rev Primer6* –Taq/antibody12 –H 2 048 Add 9ul + 1 DNA

7. Potentially SuccessfulTry Something New

8. Customers Assays 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 MgCl 2 Use hot-start with Klen taq in a complex with TaqStart antibody Use recommended primer concentration Use 15-30ng template DNA

10. Cycling Conditions Block to Capillary Major changes required for Adaptation to PCR in a Capillary System Cycling Protocol has 3 Components –Initial Denaturation (high temperature step) 1-2 minutes at 94-96 0 C –Amplification protocol-where the issues are found –Re-annealing: 94 0 C 10-20 seconds> maximum ramp to 40 0 C, hold for 1 minute

11. The Amplification Protocol 2 out of 3 are pretty easy Denaturation: 0-2 seconds at 94 0 C is widely applicable, at least 95% of all fragments 300 bp or less Annealing Temperature & Hold Duration- the biggest issue in adapting a customers assay Extension: 2-5 seconds at 72 0 C is widely applicable, at least 95% of all fragments 300 bp or less

12. Anneal Temperature Gradient Calculated Tm = 62 0 C 62 0 C 74 0 C

13. Primers bind DNA and Melt as do any other dsDNA molecule Beginning of Melt Inflection ~90 0 C End of Melt Inflection at ~92.1 0 C Tm ~91.3 0 C Difference Tm to release 0.8 0 C Tm of primer on previous slide was not 62 0 C

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 customers 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. MgCl 2 : its Influence on Product Yield and Specificity If undesired product is present on gel reducing the MgCl 2 concentration can increase specificity If product yield is not robust, increasing MgCl 2 can increase product yield

17. Increasing product yield by altering MgCl 2 concentration

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

19. Select 2mMSelect 3mM

20. LightCycler or Block-Cycler Assay:Determining a melt window for a new fragment, ramp 0.3 0 /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 customers assay to DB/HRTD chemistry?

22. Its all about the PCR