Pcrbased Methods For Telomere Length Measurement

A technique that addresses some of the limitations of Q-FISH is single telomere length analysis (STELA). Using STELA, a 20-mer noncomplementary oligonucleo-tide with a TTAGGG tail is linked to the G-rich 3' overhang of the telomere. The TTAGGG tail is then ligated to the complementary 5' strand of the telomere. PCR is performed using one primer for the linked oligo and a second primer recognizing unique subtelomeric sequence. Use of this technique requires identification of subtelomeric sequences, which has not yet been accomplished in avian species, but should be possible in chicken now that the genome is sequenced (Nakagawa et al., 2004 and references therein). Edges of telomeric DNA were identified in the draft sequence for the macrochromosomes (ICGSC, 2004, see supplementary information).

A second PCR-based technique that can be used to compare the abundance of telomere repeats is quantitative real-time PCR (Q-PCR). This technique quantifies the fold-difference between telomere-repeat copy number in an experimental sample compared to a reference DNA sample. Disadvantages of this method are that it does not determine absolute telomere length and that interstitial telomere sequences, present in avian species, will be measured as well as terminal repeats (Nakagawa et al., 2004 and references therein). This should not be a problem if telomere shortening is being measured, because the number of interstitial repeats should not change relative to terminal repeats unless dramatic genome reorganization such as a breakage-fusion-bridge cycle is occurring.

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