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Linear amplification DNA sequencing directly from single phage plaques and bacterial colonies.

Quicker ways of sequencing short DNA would facilitate both mutational analyses and long range mapping with sequence tagged sites (STS). The method of sequencing directly from plaques and colonies reported here (Fig. 1.) is a labor-saving modification of the linear amplification method using partially purified DNAs (1,2). Our method is amenable to automation and is more efficient than those requiring prior PCR amplification when only < 200 bp of sequence are needed. To sequence X, or M13 phage DNAs, a single plaque was suspended in 10 I1 water, vortexed for 5 min and incubated at 37°C for 15 min. The suspension was centrifuged at 13,000 xg, the supernatant was used for sequencing: 2 pmol of primer was 5' end-labelled with a 3-fold molar excess of [(y-32P]ATP and 5U of T4 polynucleotide kinase in 10 Al. One-fourth volume was added without purification to tubes containing: (a) 5 jiM of each dNTPs, (b) 0.5 mM ddATP, ddCTP or ddTTP, or 0.1 mM ddGTP, (c) 10 mM Tris, pH 8.3, 50 mM KCI and 1.5 mM MgCl2, (d) 2 ,Il of plaque suspension, and (e) 2.5 U of Taq (Life Technologies) or AmpliTaq (Cetus) Polymerase. The reactions were carried out in a 10 Al final volume in a Thermal Cycler (Perkin-Elmer Cetus) at 95°C, 4 min and then 30 cycles of 95°C, 1 min; 42°C-55°C (depending on primer), 2 min; and 72°C,