scispace - formally typeset
Search or ask a question

Showing papers by "Richard K. Wilson published in 1990"


Journal ArticleDOI
TL;DR: The results of these initial experiments suggested that low-pressure shearing offered a useful alternative to sonic and enzymatic DNA fragmentation methods, and additional DNA sequencing experiments using subclones produced by the low pressure-shearing method are in progress.
Abstract: Several methods have been described for random fragmentation of DNA. These methods, often used for library preparation and subcloning prior to DNA sequence analysis, include sonic treatment (1, 2), partial digestion by restriction endonucleases (3) and treatment with DNase I in the presence of manganese ions (4). While all of these methods have been used successfully to prepare random DNA fragments for further manipulation and analysis, each has difficulties and limitations. In an effort to minimize template DNA preparation tasks and simplify primerand PCR-directed DNA closure methods after an initial shotgun sequencing approach, we wished to prepare random subclones containing inserts with an average size of 4 to 6 kilobase pairs (kb). As an alternative approach, several different DNA samples were passed through a small French pressure cell at a variety of low to intermediate pressures (Figure lb). A lever device was constructed to allow controlled application of low to intermediate pressures to the cell (Figure la). The results of these initial experiments suggested that low-pressure shearing offered a useful alternative to sonic and enzymatic DNA fragmentation methods. Subsequently, regions of the Caenorhabditis elegans genome cloned in cosmid vectors were sheared using an application of 250 psi. Shearing experiments with three different C. elegans cosmid clones (insert sizes ca. 35 —42 kb) all produced essentially the same results (data not shown). The sheared cosmid DNA fragments were made flush with T4 DNA polymerase in the presence of 100 /tM dNTPs (2), and DNA fragments of the desired size range were purified by preparative agarose gel electrophoresis and subcloned in the Hindi (Sail) site of the phagemid vector pUC118. To check the efficiency of this subcloning method, 109 of these subclones were examined by standard plasmid mini-prep and agarose gel electrophoresis procedures. 101 (92%) subclones contained an insert of the expected 4 to 6 kb size range. 72 subclone DNAs were sequenced using a linear amplification method with fluorescent dye-labeled primers. Identical subclones were not observed in this analysis, and no sequence-specific shearing hot spots were detected. Additional DNA sequencing experiments using subclones produced by the low pressure-shearing method are in progress in order to determine the complete nucleotide sequence of a 100 kb region in the large cluster of C. elegans chromosome HI.

64 citations