Ligation- and PCR-Based Method for Sequencing Plasmid Inserts

摘要

Sequencing plasmid inserts frequently requires the synthesis of primers or involves subcloning steps. In order to avoid these costly and labor-demanding approaches, we have made use of a ligation- and polymerase chain reaction (PCR)-based method thatprovides high-quality DNA for sequencing. Here we describe a robust sequencing method for PCR products that reduces problems associated with DNA secondary structure and significantly streamlines the gel-loading process. The method we describe was developed to allow rapid sequencing of unknown cDNAs from Drosophila melanogaster that are expressed during early embryo-genesis. We have obtained the entire sequence of an unknown 3 0-kb cDNA cloned from a 3- to 12-h embryo library. Twelve DNA sequencing templates were produced and sequenced in one day, with an additional day necessary for running the sequencing gel and analysis. The approximate read length was 300-350 bp, providing a total of 3840 bp.