Biomedical Engineering Reference
In-Depth Information
Fig. 2. Quantification by real-time polymerase chain reaction. Shown are typical
real-time curves for amplification reactions of varying initial target concentrations
(A)
, and the log of the initial concentration plotted against the cycle number at which
the signal rises above background
(B)
as calculated by the second derivative maxi-
mum. (Adapted from
ref.
2
, with the permission of ASM Press.)
After fluorescence normalization, the shapes of the curves correlate with geno-
type. Melting domains are clearly identified as multiple melting transitions.
This closed tube technique can be used for genotyping known mutations
(
7-9
)
and for scanning of unknown mutations
(
16
)
.
Genotyping by melting curve analysis can be localized to a specific region
and made more specific by limiting the amplicon length
(
9
)
, or by using probes
of various designs (
Fig. 5
). With short amplicons, the melting rate is kept at
