Figure 1.
Schematic of PCR methods for mapping transposable elements.
A) Schematic for the inverse PCR method. Genomic DNA isolated from a fly strain containing a transposable element is digested with an enzyme that cuts within the transposon. These fragments are circularized by a ligation reaction. A PCR reaction with primers designed to the transposon end and an internal sequence amplifies the flanking genomic region. This PCR product is sequenced by a nested primer. B) Schematic for the splinkerette PCR method. Genomic DNA is isolated from the fly line containing the transposable element to be mapped. The genomic DNA is digested by an appropriate enzyme that produces sticky ends. The enzyme could cut within the transposable element (similar to scheme A for iPCR) but such digestion is not necessary for the splinkerette PCR reaction. A double stranded splinkerette oligonucleotide with a stable hairpin loop and compatible sticky ends is ligated to the digested genomic DNA. This is followed by two rounds of nested PCR (‘S1’ and ‘T1’ indicate the primer pairs for the first round from splinkerette and transposon, and ‘S2’ and ‘T2’ indicate the primer pairs for the second round of PCR). This generates a PCR fragment that contains the flanking genomic DNA between the transposable element insertion site and the genomic digestion site. A third nested primer directed against the transposon (T3) is then used for a standard Sanger sequencing reaction. In this schematic, only one end of the transposable element is targeted for isolation of flanking genomic DNA. The other end can also be targeted by using different ‘T’ primer pairs specific to this other end. C) The annealed splinkerette oligonucleotide sequence is shown along with alignment of the PCR primers SPLNK#1 (S1) and SPLNK#2 (S2). The GATC sticky end is bolded.
Figure 2.
Comparison of spPCR and iPCR for mapping of P-elements in Drosophila.
A) Representative agarose gels showing 5 µl of PCR products for inverse PCR and spPCR reactions. Genomic DNA from four fly strains (white1118 serves as a negative control) were subjected to iPCR or spPCR to isolate the 5′ or 3′ flanking genomic DNA of the P-element insertion site. For spPCR, genomic DNA was digested separately with four restriction enzymes (BfuCI, BstY1, BglII, BamHI) which produce GATC sticky ends compatible with the spPCR protocol. BfuCI iPCR products are larger than BfuCI spPCR products since iPCR amplifies more P-element specific (non-flanking genomic) DNA. DNA ladder (L) units are in kB. B) Schematic of the genomic locus containing the mapped NP2559-GAL4 enhancer trap element within the micropia natural transposon. C) Schematic of the genomic loci for the mapped GH146-GAL4 and NP225-GAL4 enhancer trap elements. The cloned GH146 and NP225 enhancer regions are shown as double-headed arrows. The PCR products for the NP225-GAL4 5′P-element BstYI and BglII spPCR fragments could not be seen on an agarose gel, but reliable sequence was obtained after phosphatase/exonuclease I treatment of the PCR product (see Splinkerette Protocol S1 for details). The flanking BglII site (marked by a *) is predicted based on the largest size (∼800 bp) of sequenced spPCR products. Ci) Agarose gel showing PCR products from the diagramed primer pairs. The P-element specific T2 primer is also diagramed in Figure 1. The lanes are labeled as in A. The mdg3 transposon at this location is not in the white1118 strain. Red triangles represent P-elements (not drawn to scale). Location of restriction sites are diagramed as vertical lines color coded according to the restriction enzymes. Restriction sites within the P-element are not shown. Black bars represent genomic DNA, green bars represent genes, yellow bars represent natural transposons, and red bars represent the extent of the longest amplified iPCR or spPCR genomic DNA fragment flanking the P-element insertion site.
Table 1.
Oligonucleotides for Splinkerette PCR of Drosophila P-elements1.
Figure 3.
Splinkerette mapping of an enhancer trap within a natural transposon highlights a master regulatory region for PN expression.
A) The expression pattern of the GH146-GAL4 enhancer trap in a representative confocal projection of a whole mount Drosophila brain immunostained for a general neuropil marker (monoclonal antibody nc82) in magenta, and for mCD8 in green (which detects GAL4-dependent UAS-mCD8-GFP expression). The antennal lobe (AL), mushroom body calyx (MB) and lateral horn (LH) regions are outlined. B) A higher magnification of the antennal lobe region for the GH146-GAL4 expression pattern. Arrowheads point to the three clusters (dorsal, lateral, ventral) of cell bodies of the projection neurons (PNs). The antennal lobe is outlined. C) The expression pattern of the NP225-GAL4 enhancer trap in whole mount brain confocal projections. D) A higher magnification of the antennal lobe region for the NP225-GAL4 expression pattern. E–F) The representative expression pattern of a transgenic construct that drives GAL4 expression from the cloned GH146 enhancer region diagramed in Figure 2C. The expression pattern in PNs appears identical to the GH146-GAL4 enhancer trap line. G) Representative confocal projections of the antennal lobe of GH146-lacZ and NP225-GAL4 animals immunostained for ßgal (in red) and for mCD8 (which reports GAL4-dependent UAS-mCD8-GFP expression) in green. H) Confocal projection of the antennal lobe of GH146-lacZ and transgenic GH146-GAL4 animals. I–J) The expression pattern of transgenic flies that contain the genomic DNA near the NP225 insertion site (“NP225 enhancer”) driving GAL4 integrated into the attP2 Φ-C31 genomic site. K–L) The expression pattern of transgenic flies that contain the “NP225 enhancer” region driving GAL4 integrated into the attP86Bb Φ-C31 genomic site. Expression in the antennal lobe is from innervation from olfactory receptor neurons, and not PNs. Scale bars: 20 µm.
Table 2.
Splinkerette PCR mapping of commonly used FRT insertions.
Table 3.
PCR conditions to test for FRT insertions.