Mutation scanning - to quickly eliminate wild type (normal) sequences
Closed Tube System: High-resolution melting operates by detection of heteroduplexes (mismatched duplexes) that form after amplification of heterozygous DNA. Some of the older mutation scanning methods also operate on this principle, but high-resolution melting is the only closed-tube method and does not require processing, reagent addition, or separation after PCR. All other methods require some manipulation of the product after PCR, followed by physical separation such as electrophoresis, chromatography, or mass spectrometry (see diagram above). Not only are these older methods labor intensive, but the need to open sample contianers after PCR is a severe disadvantage, increasing the risk of contamination in future reactions because PCR products are exposed to the environment.
Data Analysis: High-resolution melting uses a heating rate of at least 0.1ºC/sec, and is usually complete in 1 to 5 min. Mutation scanning by this method depends on the melting of heteroduplexes that distort the shape of the melting curve. In order to accurately compare curve shapes, the high-termperature end of the curves are superimposed by shifting the curves along the temperature axis until they are overlaid (see Fig 2A). Because the difference between curves is small, we magnify it by plotting the subtractive difference between samples (Fig 2B). Each curve is usually subtracted point-by-point from the homozygous reference (or an average of all wild type curves analyzed). Although difference curves look similar to derivative melting curves (Fig 2C), the twoshould not be confused.
Fig 2. Above are melting curve plots useful in mutation scanning. WT: wild type homozygote; HET: heterozygous DNA (A>G single base change).
Sensitivity and specificity of high-resolution melting analysis is better than many of the conventional mutation detection methods including dHPLC. It is more sensitive than DNA sequencing when variants are present at low allelic fractions (high-resolution melting can detect down to 2% of variant DNA in the background of normal DNA, whereas DNA sequencing can only detect down to about 20% variant). Single-base changes, insertions and deletions can all be detected, as long as the PCR primers flank the variation.
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