Dye-binding/High-resolutionThermal Denaturation

PCR-what makes or breaks fragment analysis

Steven F. Dobrowolski, PhD

Why PCR is the Most Critical Issue

dsDNA binding dye indiscriminately bind dsDNA

– Specific Product, Undesired Product, and Dimer Product all incorporated dye with equal affinity

– All products contribute to the fluorescent signal

– All products contribute to the melting profile.

Take Home Message

Specific PCR is the only means to generate

meaningful DB/HRTD data

Polymerase Chain ReactionAlternative Formats

Capillary Tube, Strip, Plate

Comparing Formats

Capillary

• High surface to volume ratio

• Glass, rapid heat transfer

• Small vapor volume• Broadly adjustable

thermal ramping rate

Tube, Strip, Plate

• Low surface to volume ratio

• Plastic, slow heat transfer

• Large vapor volume• Minimally adjustable,

slow to moderate thermal ramping rate

PCR in Capillaries

• Low volume reactions, 5-10μl is the norm• Reaction chemistry is often different than the

same reaction in tube/plate. (addressed tomorrow) • Cycling conditions very different than the

equivalent reaction in a tube/plate • Adjustable ramp speed allows fine tuning of

amplification.• PCR in capillaries is often more robust than

possible in plate/tube

32 specimens

Real-time Amplification

DB/HRTD Melting Profiles

Factors that effect PCR• Effective Primer Design (don’t trust software)

• Primer Purity (salted out, cartridge, HPLC, Gel)

• Primer Concentration (how much is optimal)

• MgCl2 Concentration (how much is optimal)

• Adjuvant (KCl, glycerol, DMSO, TMSO, others)

• Genomic DNA (concentration, chemistry, salts)

• Cycling Conditions (how many, anneal temp, hold time, ramp speed)

• Enzyme, hot-start broadly recommended

Issues Specific to DB/HRTD

Fragment size (bp)

Sensitivity Specificity

< 300 100% 100%

400 – 1000 96.1% 99.4%

Total (1248 rxns) 97.8% 99.7%

Reed and Wittwer Clin Chem 2004

Situations to Avoid

• PCR for alternative systems used precisely the same way for DB/HRTD– PCR for sequencing need not be robust– PCR for paired probes using FRET and

SimpleProbe are asymmetric– PCR for TaqMan gains specificity from the probe,

thus in need not be highly specific.– PCR for RFLP need not be highly specific

because the restriction pattern is the final data.– SSCP, DGGE, have their own issues G:C clamps,

tailing with sequencing primers, etc

How to avoid looking foolish when demonstrating DB/HRTD

• Never tell a customer adding dye to existing PCR assays is all that is necessary

• Never add dye Post-PCR and expect quality profiles

• Always see a gel of the customers PCR product before using such to demonstrate the HR-1

• Always request DNA sequence characterized samples when demonstrating HR-1

• Always assay duplicates when demonstrating HR-1

• Always run a gel if duplicates are inconsistent

Steve’s DB/HRTD Mantra

It’s All About

The

PCR