TY - JOUR
T1 - Splinkerette PCR for mapping transposable elements in Drosophila
AU - Potter, Christopher J.
AU - Luo, Liqun
N1 - Copyright:
Copyright 2010 Elsevier B.V., All rights reserved.
PY - 2010/4/13
Y1 - 2010/4/13
N2 - Transposable elements (such as the P-element and piggyBac) have been used to introduce thousands of transgenic constructs into the Drosophila genome. These transgenic constructs serve many roles, from assaying gene/cell function, to controlling chromosome arm rearrangement. Knowing the precise genomic insertion site for the transposable element is often desired. This enables identification of genomic enhancer regions trapped by an enhancer trap, identification of the gene mutated by a transposon insertion, or simplifying recombination experiments. The most commonly used transgene mapping method is inverse PCR (iPCR). Although usually effective, limitations with iPCR hinder its ability to isolate flanking genomic DNA in complex genomic loci, such as those that contain natural transposons. Here we report the adaptation of the splinkerette PCR (spPCR) method for the isolation of flanking genomic DNA of any P-element or piggyBac. We report a simple and detailed protocol for spPCR. We use spPCR to 1) map a GAL4 enhancer trap located inside a natural transposon, pinpointing a master regulatory region for olfactory neuron expression in the brain; and 2) map all commonly used centromeric FRT insertion sites. The ease, efficiency, and efficacy of spPCR could make it a favored choice for the mapping of transposable element in Drosophila.
AB - Transposable elements (such as the P-element and piggyBac) have been used to introduce thousands of transgenic constructs into the Drosophila genome. These transgenic constructs serve many roles, from assaying gene/cell function, to controlling chromosome arm rearrangement. Knowing the precise genomic insertion site for the transposable element is often desired. This enables identification of genomic enhancer regions trapped by an enhancer trap, identification of the gene mutated by a transposon insertion, or simplifying recombination experiments. The most commonly used transgene mapping method is inverse PCR (iPCR). Although usually effective, limitations with iPCR hinder its ability to isolate flanking genomic DNA in complex genomic loci, such as those that contain natural transposons. Here we report the adaptation of the splinkerette PCR (spPCR) method for the isolation of flanking genomic DNA of any P-element or piggyBac. We report a simple and detailed protocol for spPCR. We use spPCR to 1) map a GAL4 enhancer trap located inside a natural transposon, pinpointing a master regulatory region for olfactory neuron expression in the brain; and 2) map all commonly used centromeric FRT insertion sites. The ease, efficiency, and efficacy of spPCR could make it a favored choice for the mapping of transposable element in Drosophila.
UR - http://www.scopus.com/inward/record.url?scp=77956299400&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=77956299400&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0010168
DO - 10.1371/journal.pone.0010168
M3 - Article
C2 - 20405015
AN - SCOPUS:77956299400
VL - 5
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 4
M1 - e10168
ER -