Biomedical Engineering Reference
In-Depth Information
4. Notes
1.
This was described earlier by Chapal et al. in another in-cell PCR procedure
(
17
)
.
Microscopic examination of the enriched CD71
+
cell population coupled to
immunomagnetic particles after fixation and permeabilization using the
IntraStain reagents showed intact cells in conjunction with the beads. However,
cells may be lost during the successive PCR steps. Centrifugation may be used
for changes of buffer and reagents
(
16
)
; however, this did not work successfully
in the current procedure. Experiments using centrifugation steps instead of
Dynabeads for shifts of buffer or washing did not result in PCR signals; further-
more, there was a tendency in increasingly severe cell loss during these centrifu-
gation steps. An alternative way to “immobilize” the fixed cells during the
different steps in the procedure may be one way to improve the method.
2.
Naturally, in one-half of all pregnancies, another fetal-specific marker DNA
sequence must be used, such as the Rhesus D gene in cases of rhesus-negative
mothers and rhesus-positive fetuses. In addition, many paternal/fetal specific
DNA polymorphisms may be usable in an allele-specific PCR setup. Further-
more, recent studies have searched for and indicated the existence of more gen-
eral specific fetal mRNA marker sequences which may be used in a
RT-PCR-modified version of the in-cell PCR procedure presented here
(
18
,
19
)
.
3.
Several different reagents have been used in the literature for fixation and perme-
abilization of cells in
in situ
or in-cell PCR protocols
(
1
,
2
)
. Initial experiments
with 1% paraformaldehyde and proteinase K treatment did not result in any posi-
tive PCR results in the current method, but this does not mean that fixation and
permeabilization reagents other than the IntraStain reagents will not work with
the method.
4.
The most pressing problem with the
in situ
or in-cell PCR technique, both in
theory and apparently sometimes in practice, is the occurrence of false-positive
results owing to diffusion of PCR products out of and/or into the fixed and
permeabilized cells
(
1
)
. This would lead to a linkage of male (or fetal) marker
sequences (in the model system the
TSPY
PCR product) and female (or maternal)
PCR products of the gene sequence of interest (in the model system
HLA-DPB1
PCR products) in the described procedure. However, this seems not to be a prob-
lem in the presented model system; only male
HLA-DPB1
polymorphisms were
detected in the mixture of male and female cells. In the literature, diffusion prob-
lems seem mainly to occur during
in situ
PCR in tissue sections, whereas the
problem seems minimized with fixed cells in suspension
(
1
,
2
)
. One way pro-
posed in the literature to reduce possible problems with diffusion is to use, e.g.,
biotin coupled to the primers or to include biotin- or digoxigenin-substituted
nucleotides. The idea is that a more “bulky” PCR product accumulated inside the
fixed cells will not diffuse out of the cells to the same extent as nonlabeled PCR
products
(
1
)
. Another modification of the procedure could be to shorten the
primer tail sequences in the first PCR and increase the length of the primers in the
two following nested PCR steps. A high annealing temperature could thus be
used in the second and third PCR, whereby no “false”
de novo
formation of linked
PCR products would occur.
