AUTOMATED SEQUENCING OF PCR-AMPLIFIED 16S RDNA ON HYDROLINK GELS

A commercially available high-performance gel solution,'Hydrolink Long Ranger', was used for direct automated sequencing of PCR-amplified double-stranded 16S rDNA. The 1.5 kb rDNA fragments were amplified directly from bacterial cell lysates and sequenced by using Tth DNA polymerase with the linear PCR sequencing protocol. Sequencing reactions wereanalyzed on an automated laser fluorescent DNA sequ encer with Hydrolink gels compared to standard polyacrylamide gels. Automated Hydrolink gel electrophoresis allowed high sequencing speeds and resulted in a marked increase in readable sequences. Upon this electrophoresis, an average of 540 bases of nucleotide was resolved by automated reading in a single sequencing run, and the resolved sequence was extended up to 600 to 700 bases by manual correction of the errors and ambiguities. This sequencing strategy is useful for the analysis of long sequences of more than 500 bases and makes it possible to obtain entire 16S rDNA sequence data for several strains within 2 days, using an automated DNA sequencer with the one dye detection system.