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High-Resolution Melting

A Bit of History...

MeltingCurve
In the 1960s, melting of double-stranded DNA was monitored by UV absorbance (hyperchromic effect). Analysis required µg amounts of DNA, and often took hours to complete, while samples were slowly heated at rates of 0.1-1.0 ºC/min.  Contemporary DNA melting analysis uses fluorescence, and because it is a more sensitive method, it needs only ng amounts of DNA (amounts easily prepared by PCR).   Fluorescent DNA melting analysis became popular with the 1997 advent of the real-time PCR instrument LightCycler®. Capillary sample formats and small sample volumes allowed better temperature control and fast melting rates of 0.1-1.0ºC/sec, shortening the melting time to a few minutes.  The fluorescence indicator used back in 1997 was the dye SYBR® Green I, a sensitive, convenient dye for real-time monitoring of amplification and melting of amplified products.

High-resolution melting analysis is a new method introduced in 2003. Similar to the LightCycler, this too was a result of collaboration between our lab at the University of Utah and Idaho Technology, Inc (Salt Lake City, UT).  The method uses high data-density acquisition, and detects small sequence differences in PCR fragments, just by direct melting. Melting curves can now be used for mutation scanning, sequence matching, and mutliplex genotyping - analyses that traditionally required processing of PCR products by electrophoresis or other non-homogeneous means. Because of its speed and simplicity, popularity of this method is growing.

Two inventions were key to high-resolution melting:  1) high-resolution instruments, and 2) novel saturation dyes. To read more, please follow the colored tabs in the navigation bar (top of page) ..



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Key references Contact us to receive reprints.
  1. First paper: Clinical Chemistry full text
  2. Book chapter: High-Resolution Melting Analysis for Scanning & Genoytyping (Tevfik)
  3. Publication by application go to our list
  4. Wittwer Lab updated publication on hi-res melting (via PubMed)
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