Telometric: a tool providing simplified, reproducible measurements of telomeric DNA from constant field agarose gels.

Cellular proliferation must be closely regulated by an organism to maintain health because cancers arise primarily through the uncontrolled growth of specific cells. Proliferation is controlled at least in part by telomere integrity. Telomeres, structural elements that confer stability at the end of chromosomes, contain 7–10 kb tandem repeats of 5′-TTAGGG-3′ in humans (6) and associated sequence-specific DNA binding proteins. DNA replication contributes to loss of telomeric DNA because typical DNA polymerases are unidirectional and require primers (8,10). Variation in telomeric length exists between individuals, between chromosomes, and between cells within an individual. This heterogeneity leads to difficulties in making a reproducible measurement of size distribution that is representative of the population. Such measurements help to assess the effect of tumorigenesis on telomeres, allowing the determination of the effect of drugs on tumors and the effect of potential chemopreventive agents. To simplify estimates of the size of telomeric repeats, we needed to improve the speed and accuracy of measurements of the distributions of DNA molecular weights from agarose gels. Although other methods have been used to measure telomere length in acrylamide and pulsed-field gels (1,3) and directly in cells using fluorescent tags (9), we required an easy-to-use system for performing accurate, reproducible measurements on inexpensive constant field agarose gels. Such a system is not available, which has led many researchers to make “eyeball” measurements of the location of peak intensity. Accurate measurements are problematic because of the nonlinear migration of DNA on gels and because telomeric repeat regions hybridize multiple times to the probe (in this case, an end-labeled 24-mer), so that intensity distributions do not reflect copy number distributions. However, the correct determination of the size and distribution of the telomeric repeats requires measures on the distribution of copies, not intensities. We have developed easy-touse, freely available software to determine the distributions by copy number and calculate statistics on them. This software, Telometric, is useful for studies of telomeric regions and for measuring DNA distributions in other contexts, such as cDNA library construction. It can be freely downloaded from the BioTechniques Software Library (www. BioTechniques.com). The software is an NIH Image macro that analyzes an autoradiograph of an agarose gel hybridized with telomeric probe. The chromosomal DNA is prepared in standard fashion. Briefly, restriction digestion of phenol/chloroform-extracted genomic DNA is carried out with HinfI and RsaI. Following digestion, DNA concentrations are determined using the fluorescent DNA quantitation kit and VersaFluor fluorometer (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer’s recommendations, and equivalent amounts of DNA are loaded per lane, ranging from a minimum of 1.0 μg/lane to a maximum of 5.0 μg/lane, on 0.7% agarose gels. Following electrophoresis, the DNA is transferred to Hybond N membranes (5). The membrane is hybridized with end-labeled oligonucleotides complementary to the telomeric repeats, 5′-TTAGGG-3′ and 5′-CCCTAA-3′, as described previously (2). The autoradiograph is scanned and imported into NIH Image (Figure 1). It is assumed that the grayscale intensity measured at each point in an image is directly proportional to the concentration of labeled DNA, essentially ignoring non-telomeric DNA attached to telomeres, so that such regions need to be either relatively small compared to the telomeric repeat region or relatively uncommon. Obviously for very small telomeric regions (<1 kb), this assumption may break down; however, the assumption seems justified above that size as shown by the distribution curves in Figure 2 that show no tail at short telomere lengths, which non-telomeric DNA would cause. Second, it is assumed that the image is unsaturated, which is easily checked by verifying that no intensity