Recovery of DNA sequences flanking P-element insertions in Drosophila: inverse PCR and plasmid rescue.
The Drosophila melanogaster P-transposable element is a powerful and widely used research tool. Sequences flanking the P-element can be recovered and the site of insertion can be mapped to the nucleotide, to connect the genetic and physical maps and facilitate molecular analysis of the gene of interest. The Berkeley Drosophila Genome Project (BDGP) has assembled a well-characterized collection of lethal mutations induced by single P-element insertions generated by a number of laboratories. The genomic DNA sequences adjacent to these insertions have been recovered by either plasmid rescue or inverse polymerase chain reaction (PCR). The combination of a complete genomic DNA sequence and relatively fast and easy molecular methods for mapping P-element insertion sites to the nucleotide enhances the use of P-elements as tools in Drosophila research. This protocol provides detailed procedures for isolating DNA flanking P-element insertions.
Huang
Audrey M
AM
Rehm
E Jay
EJ
Rubin
Gerald M
GM
D000818:Animals; D002874:Chromosome Mapping / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D004251:DNA Transposable Elements / Q000235:genetics*; D004330:Drosophila / Q000235:genetics*; D005821:Genetic Techniques; D009154:Mutation; D009711:Nucleotides / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*
D004251:DNA Transposable Elements; D009711:Nucleotides; D004247:DNA