Table 2. Characteristics that affect the ability to distinguish homozygotes.
Characteristic and classificationnAccuracy (%)Pa
Instrument factors
    Melting protocolb<0.0005
        Recommended by manufacturer100472.7
        Ramp rate 0.1°C/s, oil overlay103862.9
    Detection mode<0.0005
        Single sample detection226878.1
        Full-plate imaging217361.8
    Acquisition mode0.114
        Continuous163168.9
        Step281071.1
    Softwarec0.002
        Custom170974.4
        Commercial161869.5
Assay factors
    Amplicon size<0.0005
        51 and 100 bp222277.7
        272 and 547 bp221962.8
    ΔTmd
        Single sample detection<0.0005
            P < 0.0561188.4
            P ≥ 0.0529764.0
        Full-plate imaging0.12
            P < 0.0522265.8
            P ≥ 0.0565571.3
    TmSD (°C)0.010
        Single sample detection240.058 (0.067)e
        Full-plate imaging160.112 (0.056)e
  • a All characteristics evaluated by χ2 analysis (except t-test for TmSD. P values < 0.05 are shown in bold.

  • b Two melting protocols were compared on 5 instruments for each product size.

  • c Commercial software provided by each instrument was compared to custom software [Gundry et al. (18), Palais and Wittwer (19)] modified to accept input from all instruments. All 10 instrument configurations and all 4 product sizes were analyzed by both manufacturer and custom software, except for the HR-1 (instrument and custom software are the same) and samples melted at 0.1 °C/s (custom software only).

  • d Tm difference between A/A and T/T obtained from the temperature peaks on negative derivative curves using custom software.

  • e Values [mean (SD)] are of TmSD, not accuracy.