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High Resolution Melting

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”.

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High Resolution Melting

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  1. High Resolution Melting What is it ? How does it work ? Jason McKinney Scientist

  2. Scanning for sequence variants • “Scanning” = SNP discovery, rare alleles • “Genotyping” = discriminate between known alleles, common mutation • Scanning based on detection of “HETERODUPLEXES”

  3. What is a “Heteroduplex” ?

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

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

  6. 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.

  7. What is a Melting Curve?

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

  9. 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

  10. HR-1 and LCGreen Instrument and Chemistry solutions

  11. What is a “Saturating” dye ?

  12. Melting with LCGreen

  13. HR-1 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 HR-1 vs. LightCycler

  14. HR-1 LightCycler Melting curves: HR-1 vs. LC

  15. 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

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