Edinburgh Napier University

  Many laboratories still find the direct sequencing of large DNA templates (i.e. lambda, cosmid and bacteriophage P1 clones) problematic. To date, only limited amounts of sequence information, which in many cases is of poor quality, can be generated from such molecules, even when using polymerase chain reaction (PCR)-based cycle sequencing protocols (Ref. 1, 3, 4, 5, 6, 7). To obtain reliable nucleotide data, it is usually necessary to subclone portions of the DNA of interest into either a high copy-number plasmid or a single-strand bacteriophage, such as M13. It would be advantageous to have a simple and rapid protocol that could reliably yield large amounts of accurate sequence information and that could be employed in conjunction with an automated fluorescence-based DNA-sequencing apparatus. Here, we describe optimized reaction parameters (template concentration, primer length and cycling conditions) for use with a commercially available dye terminator DNA sequencing kit, and show the excellent quality of the data that can be obtained from either lambda, cosmid or bacteriophage P1 DNAs. This procedure is performed in a single tube, using unmodified oligonucleotide primers, on templates that do not require extensive purification....