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

11. What is a “Saturating” dye ?

12. Melting with LCGreen

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

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

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