Rapid and Automated Cartridge-based Extraction of Leukocytes from Whole Blood for Microsatellite DNA Analysis by Capillary Electrophoresis

摘要

The human genome contains microsatellite sequences, consisting of 2- to 5-bp repeats, randomly distributed among the chromosomes. These sequences, also called short tandem repeats (STRs), have been demonstrated as important markers for disease diagnostics, genetic mapping, and human identification. The list of human hereditary diseases associated with the unusual expansion or deletion of specific microsatellite loci continues to increase. The two most understood diseases of this type are the CGG repeats in fragile-X syndrome (1) and the CAG repeats in Huntington disease (2). However, other diseases, such as cancer of the colon, head, neck, gastrointestinal track, urinary bladder, liver, lung, breast, and leukocytes [white blood cells (WBCs)], have been shown to be linked to microsatellite instability (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14).The growing importance of microsatellite markers in diagnostics has motivated us to develop rapid, automated devices to integrate sample preparation, PCR, and capillary electrophoresis (CE) analysis. The work presented here demonstrates an efficient filtration cartridge (15) to automatically process WBCs in a sample of blood for PCR amplification and CE analysis. A flow-through sample preparation procedure is used that does not require vortex-mixing, precipitation, and centrifugation, and is amenable to integration into a microfluidic circuit. TH01 , a well-characterized microsatellite locus, served as the model for this study. TH01 , a gene for human tyrosine hydroxylase, is a …