Nested deletions from a fixed site as an aid to nucleotide sequencing: an in vitro system using Tn3 transposase.
We have previously constructed a cloning/sequencing vector, with an in vivo system capable of creating nested deletions from the end of transposon Tn3, which is useful for sequencing large DNAs. Here we report an in vitro system which uses an ammonium sulfate fraction of extract from E. coli cells harboring a Tn3 transposase overproducer plasmid to generate nested deletions. A key feature of the procedure is exhaustive digestion of the reaction products with a restriction enzyme that cleaves only between the Tn3 "right" terminus and the cloned fragment. This step reduces the noise level due to mechanisms other than deletions from the Tn3 terminus, and facilitates detection and isolation of the desired deletion products. This system enables us to save at least 2 days' time when obtaining the necessary deletions compared with the in vivo system.
Morita
M
M
Laboratory of Molecular Biology, Kansai Medical University, Osaka, Japan. morita@makino.kmu.ac.jp
Umemoto
A
A
Li
Z X
ZX
Nakazono
N
N
Sugino
Y
Y
D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004268:DNA-Binding Proteins / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D017384:Sequence Deletion; D014158:Transcription, Genetic; D019895:Transposases
D004268:DNA-Binding Proteins; D004254:DNA Nucleotidyltransferases; D019895:Transposases; D004262:DNA Restriction Enzymes