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