The PCR assay for targeted integration upstream of tDNAs in S. cerevisiae. The pVIT41 donor plasmid (Lauermann et al. 1997) contains a unique oligonucleotide-binding site referred to as SSB. LTRs are shown as triangles, with the wide part of the triangle representing the U3 region. After induction on galactose medium to express the Ty1, the Ty1 transcript is translated, reverse transcribed, and the resulting cDNA is integrated into the yeast genome in either orientation. Insertion events are detected by using a primer complementary to the SSB in the Ty1 element in combination with a tDNA-specific primer. PCR products labeled with an asterisk (*) are >6 kb long and are not generated by the PCR conditions used in these experiments.
