Development of a miniaturized, automated assay for genetic testing

There is a large gap between the goals of current genomic research and the possibility for patients and families affected with rare genetic diseases to receive a simple and rapid genetic diagnosis. Miniaturization and automation of analytical procedures surely have a significant impact on diagnostic testing and analytical devices based on miniaturized platforms like Lab-on-a-chip (LoC) could provide an important contribution to the diagnosis of rare diseases. Our aim is to develop a LoC and a dedicated portable analytical instrument for genotyping, capable of carrying out fully integrated sample preparation and genotyping from small volumes of crude biosamples, such as whole blood. Here, we present a preliminary LoC system platform suitable for nucleic acid extraction from whole blood and for performing Real-time PCR to detect sequence variations on human genomic DNA. Preliminary work was focused on developing the single analytical steps including DNA extraction and assay design for allelic discrimination with Taqman probes on polymer-made chip support. At the same time a prototype of a polymer LoC and an instrument for miniaturized DNA purification and real-time PCR have been developed.\r\nFinal conditions were applied to the LoC platform for the detection of the p.Pro369Ser mutation in the human pyrin/marenostrin (MEFV) gene associated with Mediterranean fever used as a model of genetic application, allowing correct genotyping of 20 samples that were heterozygous, or wild-type for this mutation. Preliminary results show that the platform we developed is very promising for rapid identification of DNA sequence variations because it is based on cost effective and convenient polymer chip format and the prototype of device will be capable of carrying out fully integrated sample preparation and genotyping from small volumes of whole blood.