A1 Refereed original research article in a scientific journal
An efficient DNA sequencing strategy based on the bacteriophage Mu in vitro DNA transposition reaction
Authors: Haapa S, Suomalainen S, Eerikainen S, Airaksinen M, Paulin L, Savilahti H
Publisher: COLD SPRING HARBOR LAB PRESS
Publication year: 1999
Journal:: Genome Research
Journal name in source: GENOME RESEARCH
Journal acronym: GENOME RES
Volume: 9
Issue: 3
First page : 308
Last page: 315
Number of pages: 8
ISSN: 1088-9051
Abstract
highly efficient DNA sequencing strategy was developed on the basis of the bacteriophage Mu in vitro DNA transposition reaction. In the reaction, an artificial transposon with a chloramphenicol acetyltransferase [cat) gene as a selectable marker integrated into the target plasmid DNA containing a 10.3-kb mouse genomic insert to be sequenced. Bacterial clones carrying plasmids with the transposon insertions in different positions were produced by transforming transposition reaction products into Escherichia coli cells that were then selected on appropriate selection plates. Plasmids from individual clones were isolated and used as templates For DNA sequencing, each with two primers specific for the transposon sequence but reading the sequence into opposite directions, thus creating a minicontig. By combining the information from overlapping minicontigs, the sequence of the entire 10,288-bp region of mouse genome including six exons of mouse Kcc2 gene was obtained. The results indicated that the described methodology is extremely well suited For DNA sequencing projects in which considerable sequence information is on demand. In addition, massive DNA sequencing projects, including those of full genomes, are expected to benefit substantially from the Mu strategy.
highly efficient DNA sequencing strategy was developed on the basis of the bacteriophage Mu in vitro DNA transposition reaction. In the reaction, an artificial transposon with a chloramphenicol acetyltransferase [cat) gene as a selectable marker integrated into the target plasmid DNA containing a 10.3-kb mouse genomic insert to be sequenced. Bacterial clones carrying plasmids with the transposon insertions in different positions were produced by transforming transposition reaction products into Escherichia coli cells that were then selected on appropriate selection plates. Plasmids from individual clones were isolated and used as templates For DNA sequencing, each with two primers specific for the transposon sequence but reading the sequence into opposite directions, thus creating a minicontig. By combining the information from overlapping minicontigs, the sequence of the entire 10,288-bp region of mouse genome including six exons of mouse Kcc2 gene was obtained. The results indicated that the described methodology is extremely well suited For DNA sequencing projects in which considerable sequence information is on demand. In addition, massive DNA sequencing projects, including those of full genomes, are expected to benefit substantially from the Mu strategy.
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