USE OF TAGGED RANDOM HEXAMER AMPLIFICATION (TRHA) TO CLONE AND SEQUENCE MINUTE QUANTITIES OF DNA-APPLICATION TO A 180 KB PLASMID ISOLATED FROM SPHINGOMONAS F199

We have developed a novel method to clone and sequence minute quantiti es of DNA, The method was applied to sequence a 180 kb plasmid pNL1. T he first step was the production of a size distributed population of D NA molecules that were derived from the 180 kb plasmid pNL1, The first step was accomplished by a random synthesis reaction using Klenow fra gment and random hexamers tagged with a T7 primer at the primer 5'-end (T7-dN(6), 5'-GTAATACGACTCACTATAGGGCNNNNNN-3'), In the second step, K lenow-synthesized molecules were amplified by PCR using T7 primer (5'- GTAATACGACTCACTATAGGGC-3'). With a hundred nanograms starting plasmid DNA from pNL1, we were able to generate Klenow-synthesized molecules w ith sizes ranging from 28 bp to > 23 kb which were detectable on an ag arose gel, The Klenow-synthesized molecules were then used as template s for standard PCR with T7 primer, PCR products of sizes ranging from 0.3 to 1.3 kb were obtained for cloning and sequencing, From the same Klenow-synthesized molecules, we were also able to generate PCR produc ts with sizes up to 23 kb by long range PCR. A total 232.5 kb sequence s were obtained from 593 plasmid clones and over twenty putative genes were identified, Sequences from these 593 clones were assembled into 62 contigs and 99 individual sequence fragments with a total unique se quence of 86.3 kb.