Biomedical Engineering Reference
In-Depth Information
5.
MGB probes emit lower background fluorescence than dual-labeled probes. This
is mainly owing to more efficient quenching because the probes are shorter. As a
result, MGB probes have a longer observable exponential phase, and this permits
the use of a wider range for the thresholds. Dual-labeled probes may also be used,
but the measurements are slightly inferior in terms of final accuracy.
It is also important to be aware that the use of different batches of the same
probes can result in differences in measurement because of minor differences in
quality.
6.
It is highly recommended that HPLC purified primers be used, as these are not
subject to artifacts that nonpurified primers are prone to, such as the formation of
unspecific products by the presence of shorter unspecific primer fragments, which
have an adverse effect on the reaction efficiency. Nonpurified primers are also
subject to greater batch-to-batch variation than HPLC purified ones.
7.
It is very important that the Chelex resin suspension be heated prior to centrifu-
gation and separation of the DNA from the resin pellet. If not, readsorption of the
DNA by the resin can occur, thereby removing a major amount of the DNA from
the solution. This adsorption process may also lead to an imbalance of the ratio of
the two target chromosomes. It is also important that no resin is transferred to the
PCR as this will adversely affect amplification.
8.
DNA concentrations need not be quantified. The amount of DNA needed for
highly reproducible results ranges from 5 to 80 ng. The protocol will usually
produce a DNA solution in the required concentration range. Because of the lim-
ited quantity of sample material available, it should not be used for concentration
measurements. The additional working step is not necessary.
By co-amplifying a reference sample of known amount of DNA, which is the
usual practice, the concentration of the sample can be determined with the fol-
lowing equation:
concentration of sample = 2
-(∆CT)
/concentration reference sample.
9.
Any kind of genomic DNA where the chromosomal ratio is preserved can be
used. In addition to the Chelex-extracted amniotic fluids, we also tested extrac-
tions with the highly pure PCR template kit from Roche and with the QIAmp
blood kits from Qiagen. Cellular materials used included whole blood, monocytes
from blood, and cultured cells from blood and from amniotic fluid.
10.
Use of a reference sample of known quantity allows quantification, as mentioned
in
Note 8
. If the sample and a dilution of it are amplified, the sequence detection
software can determine the concentration of all samples tested with two standard
curves (one for each chromosome).
In theory, these curves would permit karyotyping of the samples. In practice,
however, the results are not good enough for karyotyping.
11.
It is a good idea to amplify both the undiluted and a diluted sample. PCR inhibi-
tors may sometimes be present in the original sample. Such inhibitors will then be
diluted during the diluting process. This added precaution makes it possible to
observe reaction efficiencies for a sample via a standard curve and, thus, reveal
the presence of inhibitors.
