High Resolution MeltingHigh Resolution Melting

What is it ?What is it ?

How does it work ?How does it work ?

Jason McKinneyJason McKinney

ScientistScientist

Scanning for sequence variantsScanning for sequence variants

• ““Scanning” = SNP discovery, rare allelesScanning” = SNP discovery, rare alleles

• ““Genotyping” = discriminate between Genotyping” = discriminate between known alleles, common mutationknown alleles, common mutation

• Scanning based on detection of Scanning based on detection of “HETERODUPLEXES”“HETERODUPLEXES”

What is a “Heteroduplex” ?What is a “Heteroduplex” ?

Scanning (continued)Scanning (continued)

• Based on detection of heteroduplexesBased on detection of heteroduplexes

• Different from fully base paired moleculesDifferent from fully base paired molecules– Mobility (SSCP, DGGE, CGGE)Mobility (SSCP, DGGE, CGGE)– Thermal stability (dHPLC)Thermal stability (dHPLC)

• Sequencing (gold standard)Sequencing (gold standard)

Effective methods, however, …Effective methods, however, …

• Time consumingTime consuming

• Labor intensiveLabor intensive

• Technical skillsTechnical skills

• Equipment / Start up costEquipment / Start up cost

• … … can we build a better “mouse trap” ?can we build a better “mouse trap” ?

Fluorescent Melting Curve analysis?Fluorescent Melting Curve analysis?

• Identify heteroduplexes based on the Identify heteroduplexes based on the change in change in fluorescentfluorescent signal as a signal as a sample is sample is thermally denatured (melted)thermally denatured (melted)

• Dye that “glows” when bound to dsDNADye that “glows” when bound to dsDNA

– Wait a second, … haven’t people tried this Wait a second, … haven’t people tried this already ?already ?• Well, … yes, … it didn’t work very well.Well, … yes, … it didn’t work very well.

What is a Melting Curve?What is a Melting Curve?

Melting with Sybr GreenMelting with Sybr Green

Lipsky, et al., Clin Chem 2001Lipsky, et al., Clin Chem 2001

WHY it did not workWHY it did not work

• 2 Primary Reasons2 Primary Reasons– (1) Chemistry(1) Chemistry

• Fluorescent dyes inhibit PCR at Fluorescent dyes inhibit PCR at ““SATURATINGSATURATING” concentrations” concentrations

– (2) Instrumentation(2) Instrumentation• Slow data acquisition rates, optically inferior Slow data acquisition rates, optically inferior

instrument, inadequate temperature controlinstrument, inadequate temperature control

HR-1 and LCGreenHR-1 and LCGreen

Instrument and Chemistry solutionsInstrument and Chemistry solutions

What is a “Saturating” dye ?What is a “Saturating” dye ?

Melting with LCGreenMelting with LCGreen

HR-1 vs. LightCyclerHR-1 vs. LightCycler

• HR-1HR-1– Data acquisition (>100 Data acquisition (>100

pts/degree @ 0.3pts/degree @ 0.300C/sec, C/sec, fast)fast)

– Temperature control Temperature control (+/- 0.05(+/- 0.0500C)C)

– Custom optics (matched Custom optics (matched to LCGreen, dedicated)to LCGreen, dedicated)

• LightCyclerLightCycler– <10 data points/degree <10 data points/degree

@ 0.1@ 0.100C/secC/sec

– +/- 1-2+/- 1-200CC– Generic optics, meant to Generic optics, meant to

be able to detect a range be able to detect a range of fluorophores, focal of fluorophores, focal distance and/or angle distance and/or angle may change between may change between samplessamples

Melting curves: HR-1 vs. LCMelting curves: HR-1 vs. LC

• HR-1HR-1 • LightCyclerLightCycler

SummarySummary

• Scanning for heteroduplexesScanning for heteroduplexes

• New twist on an old gameNew twist on an old game

• Melting curve, sequence of eventsMelting curve, sequence of events

• HR-1, LCGreen, … solves 2 issuesHR-1, LCGreen, … solves 2 issues– Saturating dyeSaturating dye– Data acquisitionData acquisition

• Scanning is NOT GenotypingScanning is NOT Genotyping