Barry L. Karger, Emanuel Carrilho, Jan Berka, Marie Ruiz-Martinez[1], Frantisek Foret, Steve Carson and Arthur Miller
Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA 02115.
The goal of this research is to construct a robust, general purpose multi-capillary instrument for high throughput DNA sequencing and analysis using replaceable polymer matrices. The instrument design is based on no moving parts and full illumination in the detection region in order to operate each column with the fastest speeds. The laser light is split into individual beams for constant irradiation of the column. The emitted fluorescence light is collected by a wide angle lens and thence on a CCD for specific dye determination. A significant effort has been conducted on column design. For example, highly stable hydrophilic wall coatings have been developed for long term column use. Secondly, the linear polyacrylamide polymer solution has been optimized in terms of polymer molecular weight and concentration for sequencing at least 500 - 600 bases in roughly a one-hour run. Furthermore, procedures have been developed for highly reproducible large scale polymerization procedures, necessary for rugged operation. The role of column temperature has also been carefully examined to increase separation speed and reduce band compressions. These studies have been coupled with appropriate sample clean-up procedures for multi-capillary operation. In addition, base-calling software routines are being developed based on digital communication procedures for sequence readings, including numerical estimates of base confidences. We will report on our latest advances, for a variety of sequencing strategies, including primer walking with short oligonucleotide primers.
*Supported by a grant from the Director, Office of Energy Research, Office of Health and Environmental Research of the U.S. Department of Energy under contract DE-FG-9060985.
[1] DOE Human Genome Distinguished Postdoctoral Fellow.