A transmission imaging spectrograph and microfabricated channel system forDNA analysis

In this paper we present the development of a DNA analysis system using a m icrofabricated channel device and a novel transmission imaging spectrograph which can be efficiently incorporated into a high throughput genomics faci lity for both sizing and sequencing of DNA fragments. The device contains 4 8 channels etched on a glass substrate. The channels are sealed with a flat glass plate which also provides a series of apertures for sample loading a nd contact with buffer reservoirs. Samples can be easily loaded in volumes up to 640 nL without band broadening because of an efficient electrokinetic stacking at the electrophoresis channel entrance. The system uses a dual l aser excitation source and a highly sensitive charge-coupled device (CCD) d etector allowing for simultaneous detection of many fluorescent dyes. The s ieving matrices for the separation of single-stranded DNA fragments are pol ymerized in situ in denaturing buffer systems. Examples of separation of si ngle-stranded DNA fragments up to 500 bases in length are shown, including accurate sizing of GeneCalling(R) fragments, and sequencing samples prepare d with a reduced amount of dye terminators. An increase in sample throughpu t has been achieved by color multiplexing.