An inverse PCR technique to rapidly isolate the flanking DNA of dictyostelium insertion mutants.
Restriction enzyme mediated integration is a widely used and effective method for insertional mutagenesis in Dictyostelium discoideum. In this method, plasmid rescue is used to clone the genomic deoxyribonucleic acid (DNA) sequences that flank the insertion site. For this to be effective, it is necessary to first find a convenient restriction enzyme site within the genomic DNA. This is a time-consuming process that requires Southern blot analysis of the mutant DNA. In addition, plasmid rescue requires transformation into highly competent Escherichia coli. Problems can arise owing to unstable genomic sequences, damage to the plasmid DNA and exogenous plasmid contamination. We have established a simple and rapid polymerase chain reaction-based technique that works for all mutants and circumvents the need for Southern blot analysis and plasmid rescue.
MCR Laboratory Molecular Cell Biology and Dept. of Biology, University College London, Gower St., London WC1E 6BT, UK.
Melanie
M
Keim
Robin S B
RS
Williams
Adrian J
AJ
Harwood
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements / Q000235:genetics*; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics*; D004023:Dictyostelium / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D014170:Transformation, Genetic
D017931:DNA Primers; D004251:DNA Transposable Elements; D016054:DNA, Protozoan; D004262:DNA Restriction Enzymes