In 1977, Frederick Sanger, S. Nicklen and Alan Coulson tested chain termination on Phi X 174 DNA. They isolated restriction enzyme fragments as primers, heated them with template to for three minutes and annealed at for thirty minutes. Four extension mixtures used the Klenow fragment of DNA polymerase and radioactive dATP. Their Figure 1 used ddGTP, ddATP, ddTTP and araCTP, an alternative terminating substrate; Figure 2 used all four dideoxy types. This distinction matters when relating the original experiments to an all-ddNTP teaching model.[ref]
DNA sequencing with chain-terminating inhibitors by Frederick Sanger and others. Sanger, Nicklen and Coulson, DNA sequencing with chain-terminating inhibitors, PNAS 74 (1977), pp. 5463–5465, Sequencing Procedure and Figures 1–2
Figure 1 used a 12% acrylamide gel at for fourteen hours, read by autoradiography. Omitting the unlabeled dATP chase caused unwanted A stops across samples: a chemistry scientific control. Ordered bands supported local nucleotide sequences; comparisons could revise provisional assignments. Weak T bands, unresolved long runs and structure-dependent migration limited interpretation. The paper's observations are evidence from particular preparations, not a guarantee that every endpoint is resolved.[ref]
DNA sequencing with chain-terminating inhibitors by Frederick Sanger and others. Sanger, Nicklen and Coulson (1977), p. 5464, chase-omission observation and Figure 1 caption; p. 5466, Results: weak T bands, unresolved runs, sequence comparisons and structure-dependent migration
Articles by others on the same topic
There are currently no matching articles.