high resolution melting
DESCRIPTION
High Resolution Melting. What is it ? How does it work ?. Jason McKinney Scientist. Scanning for sequence variants. “Scanning” = SNP discovery, rare alleles “Genotyping” = discriminate between known alleles, common mutation Scanning based on detection of “HETERODUPLEXES”. - PowerPoint PPT PresentationTRANSCRIPT
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