The labeling efficiency of human telomeres is increased by double-strand PRINS.
Telomeres are composed of tandem repeated sequences, TTAGGG, that can be detected either by fluorescence in situ hybridization (FISH), more efficiently by using a peptide nucleic acid (PNA) probe, or by the primed in situ (PRINS) technique. However, the efficiency of human telomere labeling using PRINS is somewhat lower than the efficiency using PNA-FISH. To solve this problem, we developed a double-strand PRINS technique, which uses two primers, (TTAGGG)(7) and (CCCTAA)(7), to label both forward and reverse telomeric DNA strands. A total of 120 lymphocyte metaphases obtained from three normal adults were scored to evaluate the labeling efficiency based upon the telomere signal frequency present in chromatid ends and chromosome arms. As a comparison, 30 metaphases from the same three individuals were evaluated using PNA-FISH. The average labeling efficiency of PRINS was increased to a level very close to that obtained with PNA-FISH. Therefore, we demonstrated that the low labeling efficiency of human telomeres with regular PRINS was likely caused by uneven annealing of primers at the relatively short human telomere sequences, resulting in some telomere sites with very weak or absent labeling. We suggest that the present double-strand labeling protocol is critical to maximize the labeling efficiency of the human telomere sequence when using the PRINS technique.
Yan
Ju
J
Department of Pediatrics, Faculty of Medicine, Université de Sherbrooke, 3001 12e Avenue Nord, J1H 5N4, Sherbrooke, QC, Canada. ju.yan@usherbrooke.ca
Chen
Bing-Zhen
BZ
Bouchard
Eric F
EF
Drouin
Régen
R
D000328:Adult; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D008856:Microscopy, Fluorescence; D020401:Primed In Situ Labeling / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D016615:Telomere / Q000737:chemistry* / Q000648:ultrastructure; D013696:Temperature
D017931:DNA Primers; D007202:Indicators and Reagents