Techniques for assessing telomere length: A methodological review

Abstract

Telomeres are located at the ends of chromosomes and have specific sequences with a distinctive structure that safeguards genes. They possess capping structures that protect chromosome ends from fusion events and ensure chromosome stability. Telomeres shorten in length during each cycle of cell division. When this length reaches a certain threshold, it can lead to genomic instability, thus being implicated in various diseases, including cancer and neurodegenerative disorders. The possibility of telomeres serving as a biomarker for aging and age-related disease is being explored, and their significance is still under study. This is because post-mitotic cells, which are mature cells that do not undergo mitosis, do not experience telomere shortening due to age. Instead, other causes, for example, exposure to oxidative stress, can directly damage the telomeres, causing genomic instability. Nonetheless, a general agreement has been established that measuring telomere length offers valuable insights and forms a crucial foundation for analyzing gene expression and epigenetic data. Numerous approaches have been developed to accurately measure telomere lengths. In this review, we summarize various methods and their advantages and limitations for assessing telomere length.

Keywords: Genetic Techniques; Sequence Analysis; Southern Blotting; Telomere Lengths.

Fig. 1

Schematic illustrations and examples of telomere lengths measurement methodsA. TRF: DNA is digested using restriction enzymes that do not recognize telomere sequences. Gel electrophoresis is then performed, followed by blotting onto a membrane. Abbreviations: kb: kilobase pairs; MWM: Molecular weight markersB. Q-PCR: The Q-PCR method compares the telomere length (T) to a single-copy gene (S) and represents it relative to each other, with the results expressed as a T/S ratio. We illustrate Q-PCR results from both long and short telomeres. In samples with long telomeres, the peak appears first, whereas in samples with short telomeres, the peak appears later.Abbreviations: S: Single-copy gene; T: Telomere lengthC. Q-FISH: PNA probe with a fluorescent dye is used to hybridize to the telomeric DNA repeats in cells. The PNA bound to telomeres can be observed through fluorescence microscopy. The fluorescence intensity is proportional to the telomere length. By quantifying the fluorescence signals, researchers can determine the relative telomere length of individual chromosomes.Abbreviations: PNA: Peptide nucleic acidD. Flow-FISH: Fluorescently labeled PNA probes specific to telomere repeats are hybridized to the telomeric DNA in cells. These cells are then passed through a flow cytometer, where the fluorescence intensity of each cell is measured. This intensity corresponds to the telomere length, allowing researchers to assess the distribution of telomere lengths in a cell population.Abbreviations: PNA: Peptide nucleic acidE. Southern and PCR base methods.STELA: Tagging telorette at the telomere end, and then the restriction enzyme action proceeds. PCR is performed on chromosomes known in the sub-telomere sequence to perform gel loading and Southern blotting.U-STELA: Tagging telorette at the telomere end, and then the restriction enzyme action proceeds. Attach binding adapters to the cleaved portion with restriction enzymes. PCR is advanced using restriction enzyme site adapter and Telorette.TeSLA: Tagging is performed at the end of the telomere using TeSLA-T. Enzyme digestion and dephosphorylation are performed. A TeSLA adapter that can be complementary to the dephosphorylation part as a white circle is attached. PCR was performed using TeSLA-T and TeSLA adapters.Abbreviations: kb: kilobase pairs; MWM: Molecular weight markers.

Fig. 2

Timeline of telomere lengths measurement methods.

Fig. 3

Classification of telomere length measurement methods based on telomere lengths, experiment time, and low/high throughput.Shapes and colors indicate the types of telomere length: Blue circles represent methods that measure average telomere length. Red triangles represent methods that can measure the shortest telomere.