Marie C. Ruiz-Martinez

A transmission imaging spectrograph and microfabricated channel system for DNA analysis.

In this paper we present the development of a DNA analysis system using a microfabricated channel device and a novel transmission imaging spectrograph which can be efficiently incorporated into a high throughput genomics facility for both sizing and sequencing of DNA fragments. The device contains 48 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 and 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 laser excitation source and a highly sensitive charge‐coupled device (CCD) detector allowing for simultaneous detection of many fluorescent dyes. The sieving matrices for the separation of single‐stranded DNA fragments are polymerized in situ in denaturing buffer systems. Examples of separation of single‐stranded DNA fragments up to 500 bases in length are shown, including accurate sizing of GeneCalling® fragments, and sequencing samples prepared with a reduced amount of dye terminators. An increase in sample throughput has been achieved by color multiplexing.

Factors to be considered for robust high-throughput automated DNA sequencing using a multiple-capillary array instrument

The overall goal of our program is to develop a robust, high throughput, fully automated DNA sequencing instrument based on replaceable polymer solutions using a multicapillary array. Significant effort has already been devoted to column and polymer chemistry in order to obtain long read lengths per run in fast analysis time. In this paper we report on progress in instrument considerations and data processing software. A simple instrument design, based on no moving parts for continuous illumination of the capillaries and detection of the fluorescent light was used for this study. Our polymer solution replacement system with the permanent connection between the buffer/chamber manifold and capillary columns on the detector side is designed to prevent the trapping of air bubbles during matrix solution replacement. A special construction of a column-electrode couple on the injection side precludes air trapping during sample injection from small sample volumes. Our in-house software now features the significant reduction of the crosstalk signal from neighbor columns, which may be a potential problem in densely packed large capillary array sequencers.