Long PCR-based technique for detection of transposon insertions in and around cloned genes of Drosophila melanogaster.
A technique to detect a transposable element insertion greater than 5 kb away from a given gene-specific site is described. PCR is performed on genomic DNA isolated from a pool containing one heterozygous mutant fly, carrying an amplifiable allele, within a pool of 100 flies with no amplifiable sequences. A model procedure for optimizing PCR conditions and a test for primer ability to amplify sequences greater than 5 kb in length from very low dilutions of mutated sequences within non-amplifiable wild-type genomic DNA are described. The optimal annealing temperature range is shown to be as narrow as a 2 degrees C interval. Careful primer design is critical to the success of detection. Under the conditions developed, we detected many local transposable element hopping events, averaging about 3-4 per 50 flies, with the size of the PCR products being in the range of 100-6000 bp. In some cases, even larger (up to 8000 bp) bands were detected.
Gordadze
A V
AV
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205-7199, USA. gordadze@biomed.uams.edu
Benes
H
H
D000818:Animals; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D004331:Drosophila melanogaster / Q000235:genetics*; D017344:Genes, Insect / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004251:DNA Transposable Elements