Biomedical Engineering Reference
In-Depth Information
Fig. 1. Real-time monitoring during amplification and melting analysis. The bot-
tom panel shows a typical rapid-cycle temperature profile that is followed by a tem-
perature ramp for melting analysis. When the fluorescent signal is monitored during
amplification once each cycle (dotted lines), it provides information on the presence or
absence of specific target sequences and allows quantification of the target. When the
fluorescent signal is monitored continuously through the melting phase (shaded area),
it can provide information that verifies target identification, or establishes genotype.
(Adapted from ref. 2 , with the permission of ASM Press.)
The dynamic nature of real-time PCR and melting analysis simplifies detec-
tion, quantification, and genotyping. Fluorescent molecules are monitored with
an optical thermocycler that provides fluorescent excitation and quantification
of the fluorescent emission. The fluorophores may be covalently linked to an
oligonucleotide to form a labeled primer or probe, or may be free molecules
that bind to double stranded DNA. Many different designs are possible, the
common feature being that they must exhibit a change in fluorescence during
PCR so that product accumulation can be monitored.
The concentration of initial template in an optimized real-time PCR reaction
determines how many cycles are necessary before the fluorescence rises. There
is a direct analogy between the in vitro amplification of DNA by PCR and the
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