Real-time PCR for prenatal and preimplantation genetic diagnosis of monogenic diseases

The provision of prenatal diagnosis requires the highest standards in laboratory practice to ensure an accurate result. In preimplantation genetic diagnosis protocols additionally have to address the need to achieve an accurate result from 1 to 2 cells within a limited time. Emerging protocols of “non-invasive” prenatal diagnosis, which are based on analysis of free fetal DNA in the circulation of the pregnant mother, also have to achieve a result from a limited quantity of fetal DNA against a high background of maternal free DNA. Real-time PCR uses fluorescent probes or dyes and dedicated instruments to monitor the accumulation of amplicons produced throughout the progress of a PCR reaction. Real-time PCR can be used for quantitative or qualitative evaluation of PCR products and is ideally suited for analysis of nucleotide sequence variations (point mutations) and gene dosage changes (locus deletions or insertions/duplications) that cause human monogenic diseases. Real-time PCR offers a means for more rapid and potentially higher throughput assays, without compromising accuracy and has several advantages over end-point PCR analysis, including the elimination of post-PCR processing steps and a wide dynamic range of detection with a high degree of sensitivity. This review will focus on real-time PCR protocols that are suitable for genotyping monogenic diseases with particular emphasis on applications to prenatal diagnosis, non-invasive prenatal diagnosis and preimplantation genetic diagnosis.