TRE5-A retrotransposition profiling reveals putative RNA polymerase III transcription complex binding sites on the Dictyostelium extrachromosomal rDNA element
Fig 2
Outline of TRE5-Absr retrotransposition profiling.
Note that TRE5-A always integrates in an orientation-specific manner with the 5’ end of the retrotransposon facing the 5’ end of the targeted tRNA gene. LAM-PCR was performed on genomic DNA prepared from a pool of blasticidin-resistant clones. The 5'-biotinylated primer bound selectively to the codon-adapted ORF1 gene of the TRE5-Absr element and did not recognize ORF1 genes of endogenous TRE5-A elements. The resulting linear, single-stranded LAM-PCR products were immobilized on streptavidin beads and washed extensively. To perform profiling of TRE5-Absr insertions at tRNA genes, exponential PCRs were performed in parallel reactions with primers specific for selected tRNA gene families (“tDNA primer library”). To profile TRE5-Absr insertions at any position in the genome, second-strand synthesis was initiated with a random hexamer primer linked to a unique adapter oligonucleotide (adapter-N6). Next, exponential PCR was performed with the adapter primer and an ORF1-specific primer to yield an “adapter primer library”. SD, splice donor site; SA, splice acceptor site.