Biomedical Engineering Reference
In-Depth Information
allow quantification of many transcripts from one reverse transcription may be
preferred
(
20
)
.
8.
Inappropriate annealing and extension can occur before PCR begins. Hot Start
techniques delay the activation of the polymerase until high temperatures are
reached. There are three common types of Hot Start methods: physical separa-
tion, antibodies, and heat-labile structural inactivation of the polymerase.
9.
If the template is genomic DNA and has not been previously denatured, an initial
denaturation of 10 s at 95°C is more than adequate. The only exception is when
heat-activated polymerases are used, in which case the instructions from the
manufacturer should be followed.
For each cycle, there is no advantage to denaturation times greater than “0” s;
that is, the denaturation temperature does not need to be held. Again, the only
possible exception is when heat-activated polymerases are used. The fact that
most PCR protocols use denaturation times of 5-60 s only reflects the poor heat
transfer of conventional instruments
(
17
)
. Melting analysis with SYBR Green I
can be used to determine the product T
m
and establish necessary denaturation tem-
peratures.
Optimal annealing temperatures depend on the T
m
of the primers, but usually range
from 50 to 60°C for 20-mers with 3 m
M
MgCl
2
. With primer concentrations of
0.5
M
, annealing is rapid and “0”-s holds can often be used. Specificity improves
as the annealing time is decreased
(
21
)
.
Extension times and temperatures depend on the target amplified. If the target is
AT-rich, extension temperatures of less than 70°C may be required, whereas
extension of GC-rich targets is faster at higher temperatures (approaching 80°C).
For most targets, extension temperatures of 70-74°C work well and are most
commonly used. Extension times depend on the length of the target. For products
less than 100 bp, extension times of “0” s are adequate, even with rapid-cycling
instruments. Products less than 200 bp should require no more than 10 s of exten-
sion. An extension time of 15-20 s may be required for products up to 500 bp,
whereas 30-60 s will more often amplify a 1000-bp segment.
Transition rates are usually programmed at 20°C/s, although a slower rate
(1-2°C/s) between annealing and extension can improve yield in some cases. In
real-time PCR (as well as in conventional PCR) there is no reason for a long final
extension.
Before melting analysis, the products should be denatured (95°C for 0 s) and
rapidly cooled (-20°C/s) to 10°C below the lowest expected melting transition.
Melting analysis on the LightCycler can be performed immediately. Alterna-
tively, the samples can be stored at 4-25°C for at least 24 h before analysis on
separate (e.g., high-resolution) instruments.
µ
10.
High-resolution fluorescent melting instrumentation
(
7-10
)
can be obtained
through Idaho Technology (HR-1 and LightScanner).
References
1. Saiki, R. K., Gelfand, D. H., Stoffel, S., et al.(1988) Primer-directed enzymatic ampli-
fication of DNA with a thermostable DNA polymerase.
Science
239,
487-491.
