Biomedical Engineering Reference
In-Depth Information
3
Real-Time Polymerase Chain Reaction
and Melting Curve Analysis
Robert J. Pryor and Carl T. Wittwer
Summary
Monitoring polymerase chain reaction (PCR) once each cycle is a powerful method
to detect and quantify the presence of nucleic acid sequences and has become known as
“real-time” PCR. Absolute quantification of initial template copy number can be
obtained, although quantification relative to a control sample or second sequence is often
adequate. Melting analysis following PCR monitors duplex hybridization as the tem-
perature is changed and is a simple method for sequence verification and genotyping.
Melting analysis is often conveniently performed immediately after PCR in the same
reaction tube. The fluorescence of either DNA dyes that are specific to double-strands or
fluorescently labeled oligonucleotide probes can be monitored for both real-time quanti-
fication and melting analysis. When used together with rapid temperature control, these
methods allow amplification and genotyping in less than a half hour.
Key Words:
Polymerase chain reaction (PCR); real-time PCR; melting curve analy-
sis; fluorescent genotyping; nucleic acid quantification.
1. Introduction
Conventional polymerase chain reaction (PCR) requires that the product be
analyzed after the reaction has finished
(
1
)
, a process which is often referred to
as “end point” analysis. End point analysis usually requires a separation tech-
nique. For example, gels may be used to separate PCR products that are then
stained with ethidium bromide. Although quantification is possible with end
point analysis, it is not a simple task. Real-time instrumentation allows PCR
quantification and analysis during amplification. In addition, end point melt-
ing analysis is often performed in the same tube as the PCR, providing a “closed
tube” system for analysis (
Fig. 1
).
