SOME MODIFICATIONS IN THE PROCEDURE OF DIRECT SEQUENCING OF PCR AMPLIFIED 16S-RDNA

A method for the analysis of PCR-amplified 16S ribosomal (r)DNA has been developed that generates sequences of excellent quality. In order to avoid time consuming purification and cloning steps and to reduce the number of non-specific terminations that frequently occur during direct sequencing of 16S rDNA, we developed a strategy that combines the advantages of several procedures described previously for different purposes, such as purification of amplified DNA via the Prep-A-Gene method and the snap-freeze technique in the sequencing protocol. Using 40-400 ng DNA per PCR reaction the amount of PCR product was sufficient to subsequently generate a complete 16S rDNA sequence. The primary structures of these genes were determined fast and reliably, yielding sequences in which the rate of unidentified positions usually did not exceed 0.2%.