Biomedical Engineering Reference
In-Depth Information
4. Notes
1.
BSA is used with PCR in glass capillary tubes to prevent polymerase denatur-
ation on the glass surface. The grade of albumin is not critical, as long as it does
not contain any DNA of interest and it is not acetylated. Siliconized tubes can
also be used but are less convenient.
2.
Most reactions work well with 3 m
M
MgCl
2
, although a concentration range of
1-5 m
M
should be tried, along with annealing temperatures of 50-65°C, depend-
ing on the T
m
of the primers. For very high-T
m
products, additives such as dimeth-
ylsulfoxide (DMSO), formamide, or glycerol may be necessary for adequate
denaturation.
3.
Any native or engineered heat-stable polymerase can be used, although their
extension rates may differ. Heat-activated polymerases require time for activa-
tion and increase the time required for PCR. 5'-exonuclease activity is required
for some probe systems that depend on probe hydrolysis. Some 3'-exonuclease
activity is useful for amplification of products longer than 1 kb for removal of
incorporation errors.
4.
The first dye used in real-time PCR was ethidium bromide
(
18
)
, an intercalating
dye that binds between the bases of dsDNA. SYBR Green I was introduced to
real-time PCR in 1997
(
19
)
and is the dye most widely used today. SYBR Green I
fluorescence is greater than that of ethidium bromide
(
2
)
and it can be viewed in
the same channel as fluorescein, a common probe label. SYBR Green I is avail-
able as a 10,000X solution in DMSO from the manufacturer (Molecular Probes).
Positive displacement pipets are necessary in order to accurately pipet small vol-
umes of the stock solution in DMSO. This measurement is critical, because
whereas a 1X solution gives maximal signal, a 2X solution completely inhibits
PCR. A new class of “saturating” dyes that are not as prone to PCR inhibition has
recently been developed (LCGreen, Idaho Technology) and enables high-resolu-
tion genotyping and scanning techniques.
5.
Many different probes can be used in real-time PCR. They can be divided into
two classes based on their mechanisms of action: hybridization and hydrolysis.
The fluorescence of hybridization probes depends on whether the probe is bound
to its target sequence or not. Fluorescence is reversible and melting curves can be
obtained. Hydrolysis probes irreversibly change in fluorescence when the 5'-exo-
nuclease activity of the polymerase cleaves the probe, separating the fluorescent
label from the quencher. The relative merits of different probes are vigorously
debated and adequately covered in commercial literature.
6.
A DNA solution of 50 ng/
g/mL) has an absorbance of 1.0 at 260 nm.
This is a convenient 10X concentration of human genomic DNA for PCR.
µ
L (50
µ
7.
Reverse transcription may be performed in the same solution as PCR (one-step)
or separately (two-step). One-step reactions may use a bifunctional enzyme that
accepts both RNA and DNA as templates, or two specific enzymes. One-step
reactions use PCR primers for reverse transcription and are common in clinical
assays, in which simplicity is paramount. In the research setting, two-step reac-
tions that reverse-transcribe all mRNA with poly dT or random hexamers and
