Closed-tube mutation scanning is perhaps the most important application of high-resolution melting analysis. Mutation scanning techniques detect any heterozygous sequence change between the PCR primers. Conventional methods of mutation scanning require downstream processing, a separation step after PCR, or both. In contrast, mutation scanning by melting is performed in the same tube as PCR without any modification to the amplification except the addition of a saturating dye to the reaction prior to PCR. Like SYBR Green® I, LCGreen™ dyes raise the Tm of primers and the PCR product by a few °C, so some re-optimization of PCR temperatures may be necessary. If high melting temperatures are a problem, DMSO (5-10%) can be added without adverse affect to the melting analysis. This is often useful in regions of high GC content.
Primers are chosen to bracket the analysis region, often entire exons including adjacent intron sequence critical to mRNA processing. The sensitivity and specificity of detection depend somewhat on amplicon size. SNP detection approaches 100% for amplicons <400 bp in length, while the sensitivity and specificity for 400-1,000 bp products is 96.1 and 99.4% respectively (Reed and Wittwer, 2004). Longer amplicons often have multiple melting transitions, although such 'domains' may be present in any amplicon depending on sequence. Scanning sensitivity does not appear to depend on the position of the variant within the amplicon or the number of melting domains.
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