Nucleic Acids Research, Vol 24, Issue 19 3778-3783, Copyright © 1996 by Oxford University Press
KK Wong, LC Stillwell, CA Dockery and JD Saffer
We have developed a novel method to clone and sequence minute quantities of
DNA. The method was applied to sequence a 180 kb plasmid pNL1. The first
step was the production of a size distributed population of DNA molecules
that were derived from the 180 kb plasmid pNL1. The first step was
accomplished by a random synthesis reaction using Klenow fragment and
random hexamers tagged with a T7 primer at the primer 5'-end (T7-dN6,
5'-GTAATACGACTCACTATAGGGCNNNNNN-3'. In the second step, Klenow-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 with
sizes ranging from 28 bp to >23 kb which were detectable on an agarose
gel. The Klenow-synthesized molecules were then used as templates 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 products
with sizes up to 23 kb by long range PCR. A total 232.5 kb sequences 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 sequence of 86.3
kb.
ARTICLES
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
Molecular Biosciences Department, Pacific Northwest National Laboratory, Richland, WA 99352, USA.
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