Biomedical Engineering Reference
In-Depth Information
in vivo proliferation of bacteria in a culture flask
(
2
)
. Initially, there is expo-
nential amplification that is not observable because the concentrations are too
low to be detected. This is followed by an observable growth phase, and finally
a plateau phase in which growth is limited by nutrient consumption or over-
crowding. The second derivative maximum (point of maximum acceleration)
of this growth curve correlates with the initial template concentration. Specifi-
cally, this fractional cycle number is inversely proportional to the log of the
initial template concentration. Such assays commonly have large dynamic
ranges (5-8 decades), precision of 5-10%, excellent sensitivity (only limited
by stochastic variation), and a specificity determined by the detection method
and PCR quality.
If required, standards can be included to provide an exact copy number for
absolute quantification. However, quantification relative to an experimental
condition (mRNA) or natural reference (diploid DNA) usually provides the
necessary information, eliminating the need for absolute standards. Sometimes
a biological reference, such as a “housekeeping gene,” is used to normalize
results between experiments.
Melting curve analysis was originally used in conjunction with real-time
PCR as presumptive identification of the target amplified
(
3
)
. SYBR Green I
dye is included in the PCR reaction at concentrations that do not inhibit ampli-
fication. After PCR, the annealed products are melted at a constant rate (usu-
ally 0.1-0.3°C/s) and the decrease in fluorescence is monitored as the strands
dissociate. Further work introduced probe melting analysis as a convenient
genotyping method, first with two labeled oligonucleotides
(
4
,
5
)
, then with
one
(
6
)
, and finally, with the development of saturating DNA dyes, unlabeled
oligonucleotides
(
7
)
. High-resolution melting techniques expand the power of
these techniques and can be performed in only 1-2 min
(
8-10
)
. When com-
bined with rapid-cycle PCR
(
11-15
)
, amplification and analysis can easily be
performed in less than 30 min.
2. Materials
2.1. Generic Reagents
For convenience and to decrease variation between reactions, it is good
practice to premix all reagents that are in common; such a mixture is often
referred to as a “master mix.” If DNA (primers, probes, and template) is not
included, these generic mixtures are stable for days at room temperature, weeks
at 4°C, and months at -20°C. If a dye is included, the mixture should be kept in
the dark. Although real-time PCR is robust and many formulations are pos-
sible, we recommend the following generic mixture. This mixture was devel-
oped for rapid-cycle PCR in glass capillaries with the LightCycler. If other
sample containers are used, the bovine serum albumin (BSA) is not necessary.
