Multiplexed automated DNA sequencing directly from single bacterial colonies.
Sample preparation has been one of the major bottlenecks for large-scale DNA sequencing projects in terms of time and cost. To improve sample throughput and to integrate the front-end tasks to capillary-array DNA sequencers, protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony is picked, lysis is accomplished in situ in the plastic sample tube using either a thermocycler or a heating block. Upon heating, the plasmids are released while chromosomal DNA and membrane proteins are denatured and precipitate to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed online DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. By the marriage of colony sequencing with the capillary array sequencer, both the front end and the back end of DNA sequencing are combined in a miniaturized format. This protocol will ultimately reduce the cost of sequencing to well below current levels.
Zhang
Y
Y
Ames Laboratory-USDOE, Ames, Iowa, USA.
Tan
H
H
Yeung
E S
ES
D004269:DNA, Bacterial / Q000737:chemistry* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA / Q000379:methods*
D004269:DNA, Bacterial