Splinkerette PCR is a powerful method for isolating the genomic regions flanking known sequences. We have applied spPCR for mapping of P-elements and piggyBac elements, but it can be easily adapted for the mapping of viral integration sites or of additional transposable elements carrying transgenes, such as minos 
, hobo 
, mariner 
, or Hermes 
. We have also shown that even the most difficult insertion sites can be mapped using spPCR. As such, transposon insertions for all GAL4 enhancer traps which are currently unmappable by iPCR could potentially be mapped by spPCR. In a large scale enhancer trap screen, 6966 insertions were analyzed by iPCR. Of those, 2536 (36%) could not be mapped by iPCR due to small flanking sequence or insertion into repetitive sequence or natural transposons (GETDB, Kyoto, Japan). Similarly, in a large scale piggyBac gene disruption screen, 11% of insertions could not be mapped by iPCR 
. Splinkerette PCR could be applied for the mapping of these insertion sites.
In our spPCR protocol, we targeted GATC sticky ends for the isolation of flanking genomic DNA. This allows for use of a number of different restriction enzymes that cut genomic DNA with different frequencies, and hence generate a predictable range of flanking genomic DNA that could be isolated. Importantly, by modifying only the TOP strand of the splinkerette oligonucleotide, different sticky ends could be targeted (see ). For example, replacing GATC with AATT will target the splinkerette to sites generated by EcoRI (G↓AATTC) digestion, and replacing GATC with GGCC will target the splinkerette to sites generated by NotI (GC↓GGCCGG) digestion. Few other changes to the spPCR protocol (besides enzyme choices) would be required. This would extend the flexibility of the spPCR method to isolate different flanking genomic segments not targeted by our current protocol.
Splinkerette PCR is also simpler to set up than iPCR. For example, mapping the 3′ end of the P-element transgenes PZ, PlacW, PGAWB, and PEP by iPCR each require a specific set of primers for the PCR reaction, and another set of primers for the sequence reaction, which in turn depends on which enzyme was used for the restriction digest (http://www.fruitfly.org/about/methods/inverse.pcr.html
). In contrast, the 3′ end of all P-elements can be mapped by using the same splinkerette primers and conditions. Given the high success rate of spPCR and its ease of use, it could be applied for standard mapping of transgenes, or for high-throughput screens.
The spPCR conditions described here can be applied to most P-elements, even if the internal components of the P-element vector are unknown. This also applies to naturally occurring P-elements. While performing control experiments using the CASPR set of spPCR primers, we made the startling discovery that our white1118
stock contained a KP element inserted on the third chromosome (data not shown). KP elements are naturally occurring P-elements that contain the same 5′ and 3′ P-element ends as the pCaSpeR based constructs, but do not contain a visible marker or a transposase gene 
. And although they cannot mobilize without the addition of exogenous transposase, if they are presented with transposase, their mobilization might cause mutations that could go unnoticed if no dramatic defects in viability or sterility result. As such, spPCR could be used to test for the presence of unexpected P-elements in one's lab stocks, especially if those stocks will be used for behavior or for transformation of transgenic constructs. Of note, the isogenized white
stocks from Bloomington Stock Center (Stock Numbers 5905 and 6326) and the Canton-S strain (Stock number 1) do not contain P-elements as determined by spPCR (data not shown).
Splinkerette PCR is similar in design to adapter-ligation PCR used in Arabidopsis
to map T-DNA insertions 
. In this technique, an annealed double stranded oligonucleotide is also ligated to digested genomic DNA. The major difference between spPCR and adapter-ligation PCR is the design of the annealing oligonucleotide: spPCR uses unmodified oligonucleotides that form a hairpin loop to reduce unwanted PCR amplifications, whereas adapter-ligation PCR used phosphorylated and C7 amino modified oligonucleotides for such a purpose. The advantage of adapter-ligation PCR is that only a single round of PCR is required, whereas spPCR often requires two rounds of nested PCR. Given the success of spPCR in the Drosophila
system, adapter-ligation PCR might also be adaptable for mapping of transposable elements. Also of note, thermal asymmetric interlaced-PCR (TAIL-PCR) has been successfully used to map many P-element and piggyBac insertions 
. However, this protocol requires three rounds of nested PCR using three different degenerate primers. Since spPCR uses unmodified oligonucleotides, it might be more cost effective when initially trying to map difficult transposon insertions.
The splinkerette protocol described here has also been streamlined and simplified from previously described mammalian protocols 
. For example, we found that ligation of the splinkerette to digested genomic DNA can be shortened to two hours at room temperature (as opposed to overnight incubations at 4°C or 16°C), and that column purification of the splinkerette ligation reaction was not necessary. As a result, splinkerette mapping can easily be performed in one to two days with reduced expense. Such changes might also be applicable to the splinkerette protocols used in mammalian systems.