Instrument Comparison for Heterozygote Scanning of Single and Double Heterozygotes: A Correction and Extension of Herrmann et al., Clin Chem 2006;52:494-503

Expanded Instrument Comparison of Amplicon DNA Melting Analysis for Mutation Scanning and Genotyping

“…These advanced techniques are already being adapted to existing real-time PCR instruments. In our prior studies (5,6 ), we evaluated the melting capabilities of 9 melting instruments found in our laboratory. These instruments varied in ability to detect homozygous mutants as well as to identify single-and double-heterozygous samples.…”

“…The displacement of the double heterozygote was so great that it was distinguishable in all cases, but accurate detection of the single heterozygote was not achieved with all instruments. Table 1 shows instrument variables and measured T m SDs compiled from this and prior studies (5,6 ). The T m SDs directly affect the ability to separate different homozygous melting curves.…”

Scanning the Cystic Fibrosis Transmembrane Conductance Regulator Gene Using High-Resolution DNA Melting Analysis

“…The sensitivity and specificity for this method, although dependent on the instrument and the dye used (27)(28)(29), appear superior to other scanning methods (14 ). The technique is attractive because it is simple, nondestructive, and amendable to high-throughput on 96-or 384-well plates.…”

Quadruplex Genotyping of F5, F2, and MTHFR Variants in a Single Closed Tube by High-Resolution Amplicon Melting

“…High-resolution methods for detecting heterozygotes (23,24 ) and homozygotes (3, 13 ) have been applied to genotyping (4,25 ) and mutation scanning (7,26,27 ). Genotyping and scanning accuracy depends on the resolution of the melting instrument and the appropriate analytical software (14,28,29 ). Instrument resolution is critical because homozygotes may differ in T m by Ͻ1°C (3, 13 ).…”

“…With temperature overlap, 4 unlinked biallelic loci could have 3 (i.e., 81) possible genotype curves. The ability to distinguish many different melting curves depends on instrument resolution and may be enhanced for automated analysis with future software; however, lower-resolution instruments are likely to require temperature-correction controls and may have difficulty distinguishing some genotypes, even with the deployment of appropriate controls (14,28,29 ).…”