The application of capillary electrophoresis for DNA polymorphism analysis.

Abstract

CE fractions may also be collected and then subjected to additional analysis. Nanoliter fractions containing size or shape fractionated DNA fragments can be collected on moving affinity membranes (125) or into sample chambers (126). The exact timing of the collection steps is achieved by determining the velocity of each individual zone measured between two detection points near the end of the capillary. The DNA samples may subsequently be identified by probe hybridization, or by PCR-linked sequencing. Capillary fractions containing metabolites and derivatives of DNA and small DNA adducts can also be sampled, and then characterized directly by highly sensitive MALDI-TOF atomic analysis (112-118) and ESI-MS (118,119). The automation and integration of PCR and CE analysis (PCR-CE) on a microchip (3-12,96) will also contribute greatly to its adoption as the analysis tool of choice. Significantly, these tools will be applied for DNA sequencing (75,108), for genome mapping (65) and genotyping (42-46), for improved certainty in disease detection (3-6,107,120) and for DNA mutation analysis (2-12,27,58). Recent improvements in the design CAE arrays and associated equipment such as the radial CAE microplate and rotary confocal signal detection system (127) overcome some of the detection limitations of linear CAE and microchip devices and allow the parallel genotyping of 96 samples in about 120 s. The integration of microreactive capillary surface assays (128) and "in-capillary" analysis will also lead to further increases in the speed and sensitivity of CE-based analysis. The recent announcement of the completion of the first draft sequence of the 90% of the entire human genome within 6 mo by Celera Genomics by sequencing random DNA fragments using several hundred ABI 3700 machines (129) illustrates the enormous efficiency realized through the automation of DNA sequencing by CAE. Sequencing was performed at an average rate of approximately 6 x 10(9) bases/yr. The CAE machines will now be employed for a concerted resequencing of genome elements to create an extremely high-density polymorphism map of the entire genome (130). This map will be based principally on single nucleotide polymorphisms, and will catapult human medicine into a new era of closely detailed genetic trait mapping to identify the genetic basis of multi-gene diseases.