Mouse telomere analysis using an optimized primed in situ (PRINS) labeling technique.
Telomeres are chromosomal elements composed of variable numbers of a TTAGGG repeated DNA sequence required for genomic stability. Telomeric length is correlated with the number of copies of this repeated DNA sequence and is an important property relevant to telomeric function. Recently, it has been demonstrated that the length of the shortest telomere, not average telomeric length, is important for cell viability and chromosomal stability. Consequently, assays permitting assessment of telomeric length are important for the analysis of genomic instability disorders. The length of individual telomeres can be analyzed using the primed in situ (PRINS) labeling reaction, which produces a labeled copy of the telomeric DNA repeats in situ. In this study, we tested different variables to optimize the PRINS reaction to enable it to be applied to the detection of mouse telomeric DNA and the study of telomeric length. The specificity, efficiency and uniformity of staining were evaluated using digital fluorescence microscopy. Labeling efficiency is dependent upon the conditions used to denature the telomeric DNA and reaction duration. Staining uniformity is increased at higher annealing and elongation temperatures as well as when a fluorescently labeled nucleotide is incorporated during the elongation step. Our results also indicate that chromosomal background staining is observed when a fluorochrome-labeled nucleotide is used as opposed to a hapten-labeled nucleotide. From this study, we conclude that an optimized PRINS technique can be reliably employed to analyze mouse telomeres and, compared with the FISH (fluorescence in situ hybridization) technique, presents advantages including greater cost efficiency and reduced processing time. These advantages may encourage wider use of the PRINS technique for quantitative evaluation of the length of individual telomeres in situ.
Lavoie
Josée
J
Division de Pathologie, Département de Biologie Médicale, Université Laval et Unité de Recherche en Génétique Humaine et Moléculaire, Centre de Recherche de l'Hôpital Saint-François d'Assise, CHUQ, 10 rue de l'Espinay, Québec, QC G1L 3L5, Canada.
Bronsard
Marc
M
Lebel
Michel
M
Drouin
Régen
R
D000818:Animals; D002842:Chromatids / Q000648:ultrastructure; D002875:Chromosomes / Q000648:ultrastructure; D005347:Fibroblasts / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008856:Microscopy, Fluorescence; D020401:Primed In Situ Labeling / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D016615:Telomere / Q000648:ultrastructure*; D013696:Temperature