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

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Presentation on theme: "Maximizing the Efficacy of Melt Profiling through Stringent PCR Optimization Steven F. Dobrowolski, PhD."— Presentation transcript:

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 Add 9ul + 1ul DNA PCR with LCGreen –10X buffer12 –10X dNTP12 –10X LCGreen12 –Fwd Primer6* –Rev Primer6* –Taq/antibody12 –H 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 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 C –Amplification protocol-where the issues are found –Re-annealing: 94 0 C 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 ~ C Tm ~ C Difference Tm to release 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 /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


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