Sequences of PCR products of Hansenula polymorpha telomeres
Genomic DNA from strain DL-1 of H. polymorpha was isolated. DNA oligonucleotides PBoli733 (5'-GCGTACGACTCACTGTAGATNNNNN-3'-O(CH2)2CH2OH) and PBoli749 (p5'-ATCTACAGTGAGTCGTACGCAA-3'-biotin) were annealed at 100 nM in 20 mM Tris-acetate, pH 7,5, 50 mM NaCl and 2 mM MgCl2 by heating to 95 oC and slow cooling to room temperature. The partial duplex (0,5 pmol) was then ligated to genomic DNA (1 *g) with T4 DNA ligase (Rapid DNA ligation kit, Thermo Scientific). PCR amplification of telomeres was performed with forward primer S12 (5'-AAGCGGCAGAGTTGGTTTCAGGATGC-3') and reverse primer PBoli745 (5'-GCGTACGACTCACTGTAGAT-3') using GC-RICH PCR System (Roche). Cycling conditions: 95oC 3 min; 95oC 0,5 min, 55oC 0,5 min, 72oC 0,5 min (30 cycles); 72oC 10 min.  The PCR fragments were sequenced on a Roche GS FLX genome sequencer using the Titanium protocol. Sequencing reads which passed the quality filters, containing specific primer sequences were extracted for further analysis.