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Maximizing the Efficacy of Melt Profiling through
Stringent PCR Optimization
Steven F. Dobrowolski, PhD
Adapting a Customer’s PCR Assay to DB/HRTD
Optimizing PCR to a Capillary System and Initial Profiling
Steven F. Dobrowolski, PhD
First Question
Is the customer currently using the LightCycler?
If yes, the job of adapting a PCR assay for melting analysis is much simpler.
Real-time PCR is Extremely Useful to Assess Amplification
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
Modifying a Typical PCR Cocktail10 reactions, 10μl volumes
• 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
Potentially Successful Try Something New
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
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
Cycling ConditionsBlock 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 94-960C– Amplification protocol-where the issues are
found– Re-annealing: 940C 10-20 seconds> maximum
ramp to 400C, hold for 1 minute
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
Anneal Temperature GradientCalculated Tm = 620C
620C 740C
Primers bind DNA and Melt as do any other dsDNA molecule
• 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
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.
Primer Concentration: a little can change a lot
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
Increasing product yield by altering MgCl2 concentration
MgCl2 and its influence on melt profiling
2 mM
3 mM
Select 2mM Select 3mM
LightCycler or Block-Cycler Assay:Determining a melt window for a new fragment, ramp 0.30/sec
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?
It’s all about the PCR