Telomere length: a review of methods for measurement

Abstract

Background: The exciting discovery that telomere shortening is associated with many health conditions and that telomere lengths can be altered in response to social and environmental exposures has underscored the need for methods to accurately and consistently quantify telomere length.

Objectives: The purpose of this article is to provide a comprehensive summary that compares and contrasts the current technologies used to assess telomere length.

Discussion: Multiple methods have been developed for the study of telomeres. These techniques include quantification of telomere length by terminal restriction fragmentation-which was one of the earliest tools used for length assessment-making it the gold standard in telomere biology. Quantitative polymerase chain reaction provides the advantage of being able to use smaller amounts of DNA, thereby making it amenable to epidemiology studies involving large numbers of people. An alternative method uses fluorescent probes to quantify not only mean telomere lengths but also chromosome-specific telomere lengths; however, the downside of this approach is that it can only be used on mitotically active cells. Additional methods that permit assessment of the length of a subset of chromosome-specific telomeres or the subset of telomeres that demonstrate shortening are also reviewed.

Conclusion: Given the increased utility for telomere assessments as a biomarker in physiological, psychological, and biobehavioral research, it is important that investigators become familiar with the methodological nuances of the various procedures used for measuring telomere length. This will ensure that they are empowered to select an optimal assessment approach to meet the needs of their study designs. Gaining a better understanding of the benefits and drawbacks of various measurement techniques is important not only in individual studies, but also to further establish the science of telomere associations with biobehavioral phenomena.

Figure 1

Schematic showing telomeric and subtelomeric regions targeted in telomere length estimation methods. (a–c) Human telomeric and subtelomeric regions are heteromorphic and vary between chromosomes (both within a person and between individuals). Telomeres (shown in black) demonstrate a continuous range of size from shorter (a), to moderate (c), to longer (b). The regions that juxtapose the telomere (shown in gray) include telomere associated repeats, degenerate (TTAGGG)_n repeats, and unique subtelomeric repeats. This area also shows variation between chromosomes (both within and between people), as illustrated here with chromosomes having long (a), short (b) or moderate (c) juxtaposed repeat regions. The TRF method results in an assessment of both the juxtaposed (subtelomeric) and true telomeric regions (indicated by brackets) with the localization of the subtelomeric region included in the measurement being variable (based primarily on the restriction enzymes used) (shown by series of solid horizontal lines). The STELA assay also includes sequences from the juxtaposed region, but the area included is specific (sequence based). Q-FISH methodologies (which include PRINS, Flow-FISH, and HT Q-FISH) use a probe specific for the telomeric region to estimate length (shown by brackets). While the probe tends to be specific for the telomeric region, it is possible that the probe could bind to a portion of the juxtaposed region (especially the degenerate repeat region). The uncertainty of inclusion of the degenerate repeats in the length estimates obtained with this methodology is indicated by a dotted line. The qPCR technique uses primers for the telomere region and a single copy gene (may be on the same chromosome as illustrated for simplicity here, or on a different chromosome).

Figure 2

Q-FISH using metaphase chromosomes to estimate telomere length. This image shows a metaphase spread (a) that has been hybridized using a PNA probe specific for the telomere (green dots at ends of chromosomes) and a PNA probe specific for the centromeric region of chromosome 2 (control probe; highlighted by arrows). The chromosomes are also stained with DAPI to visualize their banding patterns. Based on their reverse DAPI banding patterns, the chromosomes are identified and aligned into a karyogram (shown in b). Following identification of the chromosomes, the average intensity of the telomeric regions is calculated, to result in chromosome-specific and arm-specific telomere fluorescent intensity values (c). The Q-FISH method on metaphase chromosomes also allows for the recognition of telomere free ends (d). Chromosomes lacking a telomere may have an increased frequency of chromosomal rearrangements, such as ring chromosomes (d; red arrow) or fusions between chromatids from different chromosomes (white arrow). This image was prepared by C Jackson-Cook using data collected from her laboratory. Image was developed for this manuscript.