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Mutation Discovery using Hi-Res Melting™
Sponsored,vendor-submitted protocol   Sponsored by Idaho Technology    Published in BioTechniques Protocol Guide 2007 (p.43)

Hi-Res Melting curve analysis is a rapid, high-throughput, closed-tube method for mutation scanning and genotyping.

PCR-amplified products are subjected to a high-precision denatuaration process in the presence of the double-strand DNA binding dye LCGreen® Plus on the Idaho Technology LightScanner® Instrument. Nucleic acid melting is tracked by monitoring the fluorescence of the mixture across a defined temperature range, generating melting profiles that can be used to identify the presence of sequence variation within the amplicon. Single point mutations and small insertions and deletions can all be identified using Hi-Res Melting. This technique has been shown to have near 100% sensitivity and specificity when used on products of up to 400 bp in length. Hi-Res Melting curve analysis relies on the use of a novel, highly saturating, double-strand DNA binding dye LCGreen Plus developed and manufactured exclusively by Idaho Technology. LCGreen Plus dye does not interfere with amplification and can thus be included in the LightScanner Master Mix, allowing the entire scanning process to be homogeneous.

Hi-Res Melting, with the LightScanner system, offers superior performance. Derivative melting curves, illustrate the detection of heteroduplexes in the heterozygous mutant using LCGreen Plus which are not detected using SYBR® Green I as shown below.

Mutations in PCR products are detected by changes in the shape of the melting curve compared to a reference sample. The charts below are examples of SNPs identified in two exons using the LightScanner Instrument and LCGreen Plus dye. Superior reproducibility is demonstrated by the overlapping curves of duplicate samples for both the wild type and the mutant samples.

Hi-Res Melting curve analysis offers a quick and highly sensitive method for mutation scanning and genotyping. Reactions are prepared using the LightScanner Master Mix containing LCGreen Plus, primers, and DNA. After PCR amplification samples are melted on the Idaho Technology LightScanner Instrument. Analysis of melting profiles allows the user to detect single point mutations, and small insertions and deletions. Hi-Res Melting is a nondestructive process to the samples, allowing for further analysis by the user if desired.

Idaho Technology Inc. holds exclusive license to the following patent publications which relate to Hi-Res Melting:

Publication 2005-0233335: Nucleic and melting analysis with saturation dyes

Publication 2006-0019253: Amplicon melting analysis with saturation dyes

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