Splinkerette PCR


Figure 1 (click to enlarge)Figure 2 (click to enlarge)

FRT Map Positions are available here.
The splinkerette protocol is available here.

In Drosophila, the most commonly used methods for introducing a transgene into the genome is mediated by the P-element transposon (Rubin and Spradling, 1982; Spradling and Rubin, 1982) or the piggyBac transposon (Handler et al., 1999). In these approaches, the transgene to be integrated is flanked by P-element or piggyBac transposable elements ends, which can integrate the transgene into the germline in the presence of a transposase enzyme. The result is a transgene inserted into the genome flanked by transposable element ends.

It is often useful to determine the exact genomic insertion site for the transgene. An approach for mapping insertion sites is splinkerette PCR (spPCR) (Figure 1). This technique was originally developed to amplify the genomic DNA between a known restriction site and a target gene (Devon et al., 1995), and then adapted to map the insertion sites of viral integrating gene traps in the mouse genome (Horn et al., 2007). In this technique, genomic DNA is digested to yield overhanging sticky ends (Figure 1). The restriction enzyme is not required to cut within the transgene. Onto this sticky end is ligated a double stranded oligonucleotide (the splinkerette) that 1) contains a compatible sticky end, 2) contains a stable hairpin loop, and 3) is unphosphorylated (Figure 2). Two rounds of nested PCR are then performed to amplify the genomic sequence between the transposon insertion and the annealed splinkerette. This is followed by a sequencing reaction with another nested primer. The spPCR reaction remains highly efficient and specific due to the splinkerette design. Since the splinkerette oligonucleotide is not phosphorylated at its 5’ sticky end, only the bottom 3’ recessed strand of the splinkerette sticky end is ligated to the 5’ phosphorylated sticky end of digested genomic DNA. In addition, the PCR primer (‘S1’ in Figure 1) which anneals to the splinkerette only amplifies DNA that has been generated as a result of a successful first strand synthesis. As a result, the PCR reaction occurs preferentially between genomic DNA that has ligated to a splinkerette oligonucleotide. In addition, background products are reduced due to the stable hairpin loop on the splinkerette: 1) it will not ligate to genomic DNA to generate non- specific priming and 2) it reduces end-repair priming (Horn et al., 2007). Since the enzyme does not need to cut within the transgene, any restriction enzyme that produces sticky ends can be used with the appropriate splinkerette oligonucleotide. As such, larger genomic fragments flanking the transgene insertion site can be isolated.

We have adapted spPCR for the mapping of transposable elements (both P-elements and piggyBacs) in Drosophila. The spPCR protocol is simple, efficient, and highly effective. To date, almost every transgene we have attempted to map (n>500) could be mapped by spPCR. Splinkerette PCR could be applied to the mapping of transgenes which proved difficult or impossible using iPCR or plasmid rescue, or for routine mapping of transposable elements in the fly.