Biomedical Engineering Reference
In-Depth Information
3.7. Anti-Contamination Procedures
1.
Perform the sample processing, DNA extraction, PCR preparation, and real-time
PCR in separate designated work areas. Prepare the PCR reagent mix within a
laminar flow cabinet.
2.
Use aerosol-resistant pipet tips.
3.
Incorporate the use of uracil
N
-glycosylase to each PCR to eliminate potentially
contaminating amplicons from previous PCR reactions.
4. Notes
1.
SRY
is located on the Y-chromosome and therefore is a fetal-specific target for
plasma of women pregnant with male fetuses.
-globin
is present in both the fetal
and maternal genomes, and, therefore, its concentration reflects the total concen-
tration of DNA in maternal plasma. The
SRY
and
β
-globin
real-time PCR assays
are described as illustrative examples for the detection of fetal and total DNA in
maternal plasma, respectively. Other fetal targets detectable in maternal plasma
include
RHD
(
18
)
and fetal-specific mutant alleles
(
20
)
.
β
2.
Fetal DNA has been detectable in both maternal plasma and serum
(
1
,
2
,
26
)
. How-
ever, the background maternal DNA in maternal serum is 14.6 times higher than
that in paired plasma samples from corresponding subjects
(
2
)
. The excess serum
DNA is contributed by blood clotting
(
27
,
28
)
. The excess maternal background
DNA translates to a 26- and 6-fold decrease in maternal serum fetal DNA con-
centration compared with maternal plasma in first and third trimesters, respec-
tively
(
2
)
. We have further observed (unpublished) that the analysis of
fetal-specific mutations revealed much higher false-negative detection in mater-
nal serum than plasma, possibly a consequence of the reduced fractional concen-
tration of fetal DNA in maternal serum.
3.
Studies have shown that prolonged storage of unprocessed whole blood leads to
lysis of blood cells with resultant elevations in maternal DNA concentrations
(
28
,
29
)
. The effect is much more apparent in plain whole blood than anticoagu-
lated blood. Significant elevation in serum DNA concentration can be demon-
strated with a 2-h delay of blood processing
(
29
)
. However, concentration of
maternal DNA derived from plasma with EDTA as anticoagulant remains stable
for 6 h
(
30
)
. These data further support the advantage of maternal plasma over
serum for fetal DNA analysis and also suggest that maternal blood samples col-
lected into EDTA tubes should be processed within 6 h.
4.
Previous studies have revealed that the way plasma is derived from maternal
whole blood has important implications on the analysis of maternal plasma DNA
(
24
)
. It has been shown that to ensure consistent and reliable analytical results,
cellular elements should be eliminated from maternal plasma either by adopting
a two-step centrifugation protocol or filtration of plasma
(
25
,
31
)
. Contamination
of plasma with residual cellular elements could result in a fluctuant background
of maternal DNA
(
25
)
, affect the quantitative interpretation of maternal plasma
total DNA concentration
(
25
)
, and cause the inadvertent detection of fetal cells
that have persisted in maternal plasma from previous pregnancies
(
32
,
33
)
.
