High Resolution Melting

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Presentation transcript:

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”

What is a “Heteroduplex” ?

Scanning (continued) Based on detection of heteroduplexes Different from fully base paired molecules Mobility (SSCP, DGGE, CGGE) Thermal stability (dHPLC) Sequencing (gold standard)

Effective methods, however, … Time consuming Labor intensive Technical skills Equipment / Start up cost … can we build a better “mouse trap” ?

Fluorescent Melting Curve analysis? Identify heteroduplexes based on the change in fluorescent signal as a sample is thermally denatured (melted) Dye that “glows” when bound to dsDNA Wait a second, … haven’t people tried this already ? Well, … yes, … it didn’t work very well.

What is a Melting Curve?

Melting with Sybr Green Lipsky, et al., Clin Chem 2001

WHY it did not work 2 Primary Reasons (1) Chemistry Fluorescent dyes inhibit PCR at “SATURATING” concentrations (2) Instrumentation Slow data acquisition rates, optically inferior instrument, inadequate temperature control

Instrument and Chemistry solutions HR-1 and LCGreen Instrument and Chemistry solutions

What is a “Saturating” dye ?

Melting with LCGreen

HR-1 vs. LightCycler HR-1 LightCycler +/- 1-20C Data acquisition (>100 pts/degree @ 0.30C/sec, fast) Temperature control (+/- 0.050C) Custom optics (matched to LCGreen, dedicated) LightCycler <10 data points/degree @ 0.10C/sec +/- 1-20C Generic optics, meant to be able to detect a range of fluorophores, focal distance and/or angle may change between samples

Melting curves: HR-1 vs. LC LightCycler

Summary Scanning for heteroduplexes New twist on an old game Melting curve, sequence of events HR-1, LCGreen, … solves 2 issues Saturating dye Data acquisition Scanning is NOT Genotyping