DNA SEQUENCES IN THE YEAST 2μm PLASMID: SOME RECENT INNOVATIONS IN SEQUENCING TECHNIQUES

ABSTRACT A sequence of 1019 nucleotides encompassing one of the 600 base inverted repeats and non-repeated flanking regions has been determined in the type A yeast 2μ plasmid cloned in pMB9. Methods are described applying the Maxam-Gilbert sequencing procedure to 3′-end labelled fragments prepared using a T 4 -polymerase catalysed repair reaction and the application of this method to DNA fragments generated by the combined action of two restriction enzymes where it is possible to selectively label only one 3′-end. Also an alternative approach to sequencing 5′-end labelled fragments using the dideoxy-nucleotides as chain terminators is described. The application of a cloning technique into phage M-13 as a means of preparing single stranded template for sequencing by Sanger's chain termination is also discussed.