Journal: Nucleic Acids Research
Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'
Figure Lengend Snippet: Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.
Article Snippet: A typical 10 µl ligation reaction contained 250 ng of DNA fragments, 5 µM Taq I adapters, 25 mM Tris–HCl (pH 7.5), 5 mM MgCl2 , 5 mM dithiothreitol, 500 µΜ ATP, 12.5 µg/ml BSA and 200 cohesive-end units of T4 DNA ligase (New England Biolabs) and was incubated at 16°C for 12–16 h. Primary PCR was done in a 25 µl reaction volume containing 200 µM each dNTP, 40 nM each primer (Taq1-N 5′-ATGAGTCCTGACCGA, and Tn3-O2, 5′-TTAACGTGAGTTTTCGTTCCACTG), 2 mM MgSO4 and 1 U Taq DNA polymerase (Promega) in 1× PCR buffer (20 mM Tris–HCl pH 9.2, 10 mM KCl, 10 mM ammonium sulfate, 0.1% Triton X-100) and adapter-ligated DNA.
Techniques: Amplification, Sequencing, Isolation, Polymerase Chain Reaction