The correlation between relative T/S ratios measured by quantitative PCR and relative TRF lengths measured by the traditional Southern blot approach in these 95 genomic DNA samples (Fig. ) strongly supports the conclusion that the new PCR method does indeed measure relative telomere lengths.
Individuals that have the same mean length of terminal hexamer repeat array may nevertheless have very different mean TRF lengths, due to restriction site polymorphisms and/or length polymorphisms in the subtelomeric regions that determine the length of the subtelomeric portion of the TRFs. Early studies showed that the mean length of this subtelomeric portion of the TRFs in unrelated individuals ranged from 2.5 to 6 kb (reviewed in 11
, p. 958). In our dataset we estimate the average value for the mean length of the subtelomeric portion of the TRFs to be ~4.2 kb, based on the value of the y
-intercept in Figure (where T/S goes to 0); furthermore, we estimate the mean length of this non-telomeric DNA in the TRFs to vary by up to 2 kb between individuals, based on the range of mean TRF lengths we observed in individuals sharing nearly the same T/S ratio (see Fig. ). Similarly, in comparing TRF length measurements with telomeric DNA measurements by quantitative fluorescence in situ
hybridization (Q-FISH) with a fluorescein-labeled peptide nucleic acid (CCCTAA)3
probe, Hultdin et al
) estimated the mean length of the subtelomeric portion of the TRFs to be 3.2 kb, and they observed at least a 2 kb variation in the mean TRF lengths of individuals sharing nearly the same level of Q-FISH telomere signal (12
) (Fig. ).
Furthermore, we have found that the value measured for the difference in mean TRF length between two DNA samples can be greatly affected by the choice of restriction enzyme(s) used to release the terminal restriction fragments; for example, a 590 bp mean TRF length difference between two siblings following complete digestion with a combination of RsaI + HinfI decreased to only a 170 bp difference when HaeIII was used instead (R. M. Cawthon, unpublished observations). Therefore, investigators aiming to identify primary factors accounting for inter-individual variation in the mean length of the true telomeric repeat sequence may do well to avoid measuring TRF lengths and focus instead on methods that determine the relative quantities of the telomeric hexamer repeats per se.
The methods published to date for the relative quantitation of telomeric DNA per se
in cell-free DNA preparations all normalize the telomere signal to another cellular DNA signal of high copy number, either centromeric alphoid DNA (3
) or Alu repetitive DNA (5
). However, the level of inter-individual variation in the copy number of centromeric alphoid DNA and Alu DNA sequences per cell is not known; if such variation is significant, then differences between individuals in relative telomere lengths measured by these methods may be quite inaccurate. By normalizing the quantity of telomere repeats to the quantity of a single copy gene, the PCR method of telomere measurement presented here avoids this problem.
In conclusion, we have demonstrated measurement of relative average telomere lengths by quantitative PCR in a closed tube, fluorescence-based assay, using a carefully designed pair of oligonucleotide primers. In this assay the telomere signal is normalized to the signal from a single copy gene to generate a T/S ratio. The resolution of the assay (detecting differences >~11.4%) should be adequate for many genetic and epidemiological studies, since T/S values vary ~2.5 fold among age- and sex-matched individuals (data not shown). The assay is simple, rapid and readily scalable to achieve a high throughput of samples. It should prove useful in the investigation of the biology of telomeres and the roles they play in the molecular pathophysiology of multiple diseases and aging.