AUTOMATED HIGH SPEED DNA SEQUENCING

  • Ewing, Andrew (CoPI)

Project: Research project

Project Details

Description

We propose to investigate the feasibility of a novel approach to DNA sequencing involving the combination of electrophoresis in thin slab gels with a method to rapidly and sequentially sample fom an array of microvials containing Sanger sequencing reaction products. The use of thin enclosed slab gels will provide a means to carry out multiple separations in parallel using a staggered sample introduction method developed in this laboratory. Under optimum conditions, up to 100 separations can be carried out per hour. Eventually, separations providing up to 600 to 1000 bases of sequence information should result in a sequencing rate on the order of 10(5) bases per hour. A key part of this system will be coupling the slab gel to a capillary for sample introduction. The capillary will be used to electrophoretically sample sets of nested fragments from nanoliter or picoliter microvials and deposit these along the entrance of the enclosed thin slab gel. Continuously moving the capillary along the entrance to the slab gel will provide a means to carry out continuous separations over hours to days. This format represents an alternative to the multi capillary approach wherein multiple gel-based separations will be carried out across the thin slab gel. The sampling method proposed will provide a means to transfer picoliter solutions of nested fragments to the thin slab gel without the use of relatively large sample wells. This will enhance the spatial resolution across the gel permitting additional separation lanes and will permit the use of thinner gels without sample overload. Gels with thickness of 22 micromoles are planned. Improved heat dissipation in the thinner gels leads us to speculate that single-base-resolved separations will be attained for 600 up to 1000 bases. Finally, the enclosed slab-gel format will permit pressurization facilitating the use of replaceable linear gels. A library of different oligonucleotide primers of known sequence will be placed in arrays of microvials for use in sequencing reactions. Initially, primers will be chosen from segments of the M13mp18 DNA template which will be used as a model system to demonstrate the method. The 600 to 1000 bases of sequence information obtained from each reaction vial will be used to reconstruct the total sequence. As the ease of overall sequence reconstruction increases dramatically with the length of individual sequences obtained from each separation, achieving separations with single base resolution of DNA larger than 500 bases is critically important. Large fragments of DNA (10 to 50 kb) can potentially be sequenced with this methodology without digestion into smaller fragments that must be reconstructed later. It should be possible to obtain a 106 base sequence in a matter of days at a cost easily under $.50 per base.
StatusFinished
Effective start/end date9/30/957/31/99

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