Sequencing 500 kb of yeast DNA using a GATC 1500 Direct Blotting Electrophoresis System.
We have developed efficient protocols for the construction of shotgun libraries and for high-throughput DNA sequencing at low cost using the optimized GATC 1500 Direct Blotting Electrophoresis system with colorimetric detection on nylon membranes. We can now sequence entire cosmids without any primer walking at low coverage (8-fold to 9-fold). We have demonstrated the efficiency of our protocols while sequencing 500 kb of yeast DNA with the GATC 1500 system. The methods described here have allowed us to achieve the highest data rate within the framework of the European Communities yeast sequencing program. In 1993, only two people were involved in generating and submitting 156,274 bp of final sequence data. Thereof, 39,360 bp have been verified independently in other laboratories, and no sequencing error has been found.
D003124:Colorimetry; D003360:Cosmids; D004271:DNA, Fungal / Q000737:chemistry*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004586:Electrophoresis / Q000379:methods*; D015723:Gene Library; D008163:Luminescent Measurements; D008345:Manganese / Q000494:pharmacology; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000706:statistics & numerical data
D004271:DNA, Fungal; D008345:Manganese; D003850:Deoxyribonuclease I