A General Method for Detecting Rearrangements in a Bacterial Genome

An effective method was developed to monitor genome rearrangement in bacteria. The whole procedure consists of five steps. (i) Genomic DNAs of reference cells and test cells are digested with the same restriction enzyme. (ii) The DNA restriction fragments from the test cells are radioactively labele...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1989-07, Vol.86 (14), p.5507-5511
Main Authors: Au, Lo-Chun, Paul O. P. Ts'o
Format: Article
Language:English
Subjects:
Bacteria
Bacteriophage lambda - genetics
Bacteriophages
Biological and medical sciences
Blotting, Southern - methods
Chromosome Aberrations
Chromosomes, Bacterial
DNA
DNA probes
DNA, Bacterial - genetics
DNA, Bacterial - isolation & purification
Electrophoresis
Electrophoresis, Polyacrylamide Gel - methods
Enzymes
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Gels
Genes, Bacterial
Genic rearrangement. Recombination. Transposable element
Genomes
Genomics
Molecular and cellular biology
Molecular genetics
Molecular Weight
Nucleic Acid Denaturation
Restriction Mapping
Single stranded DNA
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Summary:An effective method was developed to monitor genome rearrangement in bacteria. The whole procedure consists of five steps. (i) Genomic DNAs of reference cells and test cells are digested with the same restriction enzyme. (ii) The DNA restriction fragments from the test cells are radioactively labeled. (iii) The labeled DNA fragments of test cells are mixed with unlabeled DNA fragments from reference cells that are 100- to 1000-fold in excess and the mixture is electrophoresed in an agarose gel. (iv) After electrophoresis, DNA fragments are alkali-denatured; this is followed by renaturation in situ in the gel. The labeled rearranged DNA fragments from the test cells will renature much slower, as compared with the nonrearranged fragments, since in this location of the gel these rearranged fragments do not have a counterpart in the driver DNA, which is in excess. (v) The DNA gel is electrophoresed in a second dimension perpendicular to the first dimension after renaturation. The denatured rearranged DNAs are revealed after autoradiography, since single-stranded DNA fragments have higher electrophoretic mobility than double-stranded fragments of the same sizes. This process of detection has been demonstrated in this report by using Escherichia coli HB101 as the reference strain and E. coli HB101 carrying λ phage DNA (1:1 genomic ratio) as the test strain.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.86.14.5507