Improved detection and comparative sizing of human chromosomal telomeres in situ

Abstract

Telomeric length dynamics are thought to play an important role both in the processes of cellular aging and cancer progression. We have revised the primed in situ (PRINS) labeling technique to allow an estimation of the relative length of individual telomeres. We illustrate the applicability of the approach by demonstrating different telomeric sizes not only between blood lymphocytes from a young and an old donor, but also among bone marrow cells from hematological cancer patients. In the latter case we found general variations in telomeric sizes as well as individual telomeric variations that would have escaped detection by other methods. An interesting finding was the selective expansion of a single telomere within a specific subset of cells.

In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes

“…This process enables localized allele-specific detection of repeated sequences in the genome [8][9][10][11] , but this method, too, falls short of detecting singlecopy genes from single primers, as would be needed for genotyping in situ. More recently, linear probes hybridized in situ have been extended not by replicating the target sequence, but by using separately added small circular DNA strands as templates for a localized rolling-circle amplification (RCA) 12,13 .…”

PCR-generated padlock probes distinguish homologous chromosomes through quantitative fluorescence analysis

“…This normalization method has previously been used to quantify human telomere size. 23 The normalized results clearly demonstrate that the method is reproducible and that interexperimental variations can be minimized using an internal fluorescence standard. The normalization procedure may therefore increase the number of alleles that can be discerned compared to when using relative fluorescence data.…”

Telomeric repeat organization—a comparative in situ study between man and rodent

“…For the staining of telomeric repeats, background staining from cross-hybridization and breaks in the chromosomal DNA can be reduced to a negligible level through the inclusion of one or more dideoxynucleotides (Krejci and Koch, 1998;. This is possible because the telomeric repeats contain only three of the four bases of DNA.…”