DNA Sequencing Directly from a Mixture Using Terminal-Base-Selective Primers

Abstract

DNA cloning is often used to select and amplify one DNA species from a mixture. However, the cloning process is complex and labor-intensive. We have developed a new two-step method for DNA sequencing directly from a mixture. The first is the introduction of a known oligonucleotide (common part) into the terminus of unknown DNA by ligation. The second is selective DNA sequencing using primers with two additional nucleotides at the 3′ terminus in addition to the common part (terminal-base-selective primers). The primers work only for templates on which the primers perfectly hybridized. This method was found to be effective for the Hind III digestion products of λ phage.