Biomedical Engineering Reference
In-Depth Information
quantitation of trace quantities of cell-free fetal DNA in the maternal circula-
tion. To test the feasibility of this approach, we first performed a small-scale
study involving trisomy 21, as this is the most common aneuploidy in live
births. In our study, we compared the gene dosage of a sequence on the Down's
critical region on chromosome 21 with a control locus on chromosome 12. Our
analysis indicated that by using such an approach of comparative quantitation
it was indeed possible to discern between karyotypically normal samples and
trisomic ones, provided that certain criteria concerning quantity and quality of
DNA and replicate reaction uniformity were met
(
18
)
. After successful comple-
tion of this pilot study, we have now extended this method for the simulta-
neous detection of trisomy 18 and trisomy 21. In this analysis, the two
chromosomes being interrogated are quantified relative to one another.
Future improvements would include the development of a test in which the
other common fetal aneuploidies (chromosomes 13, 16, X, Y) would be ana-
lyzed in a matrix-type assay. By using such a matrix approach, whereby the
dosage of each chromosome is measured relative to the other chromosomes, an
automatic system of “checks and balances” would be implemented, thereby
helping to reduce the potential error rate considerably.
The use of the real-time PCR assay need not be restricted solely to the detec-
tion of aneuploidies, but can also be applied for the discernment of transloca-
tions and for the distinction of these from chromosomal trisomies. This can be
achieved by amplifying sequences critical to the region translocated, such as
those located in the Down's region of chromosome 21, which should enable
the detection of those unbalanced Robertsonian translocations that occur in
approx 4% of Down syndrome cases. It must be noted, however, that the design
of the assay does not permit one to detect triploidies, because the chromosomal
balance is equal to that of a normal karyotype.
Advantages of the real-time PCR test for prenatal diagnosis are that it is
insensitive to maternal blood contamination of samples where the maternal
DNA is present at a small percentage (about 20% or less of the total DNA in
the sample). On the other hand, this test will not be able to detect low-level
chimerism. Akin to the PCR amplification of STRs, the real-time PCR test also
can be used on a wide variety of sample materials and does not require fresh or
specially stored cells, because it only requires genomic DNA. Furthermore, the
assay is readily amenable to automation, and by making use of the current real-
time PCR 96- or 384-well formats, it also facilitates high throughput. In addi-
tion, by making use of a closed system, whereby the samples are analyzed
directly in the PCR reaction vessel during the amplification and do not need to
be opened for analysis as is the case for the analysis of STRs, the assay is also
less prone to contamination.
