Biomedical Engineering Reference
In-Depth Information
Although the use of
in situ
PCR has been hampered by problems of diffusion
of PCR products and thereby false-positive results, this seems not to be a sig-
nificant problem with cells in suspension
(
1
,
2
)
. In-cell PCR has been used for
intracellular detection of human immunodeficiency virus (HIV) mRNA and
DNA
(
3
)
. In mixtures of cells in suspension with different genotypes, in-cell
PCR may be used for intracellular detection of DNA or mRNA sequences spe-
cific for one of the genotypes/population of cells
(
4
,
5
)
. The method described
here shows that it is possible to genotype a specific gene of interest derived
from one individual in a mixture of cells from two individuals. This new
method is based on in-cell PCR and is dependent on the presence of a well
characterized and specific DNA sequence or polymorphism, which must be
present only in the cells from the individual being genotyped for the gene
sequence of interest. Fetal cells, DNA, and RNA have been detected in mater-
nal blood and plasma
(
6-12
)
. The in-cell PCR method might find diagnostic
applications in detection of intracellular pathogenic sequences in subgroups of
cells and in noninvasive prenatal genetic diagnoses with the use of the few
fetal cells circulating in maternal blood. A model system of mixtures of male
and female cells will be used to illustrate the principle of in-cell PCR for
genotyping a specific polymorphic sequence in the male cells. Mixtures of cells
from two different individuals are fixed and permeabilized in suspension. After
coamplification of a DNA sequence specific for one of the individuals and the
DNA sequence to be genotyped, the two PCR products are linked together in
the fixed cells positive for both DNA sequences by complementary primer
tails and further amplification steps. In mixtures of male and female CD71-
positive cells from umbilical cord blood attached to immunomagnetic particles,
a Y-chromosome-specific sequence
(TSPY)
is linked to a polymorphic Human
Leukocyte Antigen
(HLA)-DPB1
sequence only in the male cells, leading to
the correct
HLA-DPB1
genotyping of the male by DNA sequencing of a nested,
linked
TSPY-HLA-DPB1
PCR product.
2. Materials
1.
Cells in suspension (in this model system: umbilical cord blood cells).
2
.
Genomic DNA.
3
.
Immunomagnetic beads coupled with antibodies (anti-CD71 Dynabeads [Dynal;
Norway]).
4
.
1X PBS/2% FCS: 1X phosphate-buffered saline (PBS) containing 20 mL/L fetal
calf serum (FCS).
5
.
1X PBS.
6
.
Magnetic particle concentrator.
7
.
IntraStain reagent set (Dako; Denmark).
8
.
Oligonucleotide primers.
9
.
Taq
polymerase (Life Technologies).
