Biomedical Engineering Reference
In-Depth Information
8
Use of Real-Time Polymerase Chain Reaction
for the Detection of Fetal Aneuploidies
Bernhard Zimmermann, Lisa Levett, Wolfgang Holzgreve,
and Sinuhe Hahn
Summary
With the advent of real-time polymerase chain reaction (PCR), it is now possible to
measure nucleic acid concentrations with an accuracy that was not possible only a few
years ago. Examples are the analysis of gene expression or gene duplications/losses,
where twofold differences in nucleic acid concentration have routinely been determined
with almost 100% accuracy. As our primary interest is in prenatal diagnosis, we have
investigated whether real-time PCR could be used for the diagnosis of chromosomal
anomalies, in particular the aneuploidies such as trisomy 21, where the difference in
copy number is only 50%. The feasibility of such an approach was first tested in a pilot
study, in which we were able to demonstrate that trisomy 21 samples could be detected
with 100% specificity. We have recently modified this test in order to permit the simul-
taneous analysis of trisomies 18 and 21, and have in a large scale analysis demonstrated
that our approach can be used for the highly reproducible and robust detection of only
1.5-fold differences in gene copy number.
Key Words:
Multiplex real-time PCR; relative quantification; prenatal diagnosis;
amniotic fluid; amniocentesis; trisomy 21; Down syndrome; trisomy 18; karyotype; aneu-
ploidy; genetics; DNA analysis; gene copy number; molecular probes; PCR primers; PCR
kinetics.
1. Introduction
1.1. Background
The detection of gross chromosomal abnormalities is a major focus of
prenatal diagnostics. The most common cytogenetic anomaly in live births is
trisomy 21, also known as Down syndrome. Other fetal aneuploidies frequently
detected involve chromosomes 13, 16, 18, and both sex chromosomes (X and
Y). Currently, prenatal diagnosis of genetic anomalies relies on invasive
