Telomere Terminal Restriction Fragment Analysis

First described in Harley et al., 1990, telomere terminal restriction fragment analysis establishes mean telomere length in a tissue or cell sample or percent of telomeric DNA present in one sample relative to another. To measure mean telomere length, genomic DNA is first digested with a restriction enzyme or a cocktail of restriction enzymes followed by electrophoretic separation through an agarose gel. It is essential that DNA concentration be equivalent in each lane. The gel is Southern blotted and hybridized to a TTAGGG(n) probe labeled with a radionuclide or a fluorochrome producing a smear of fragments. Densitometry readings taken at a number of locations along the smear are summed and averaged. Mean telomere length is defined as (ODi)/ (ODi/Li) where ODi is the densitometer output and Li is the length of the DNA at position i. Sums are calculated over the range of lengths covered by the smear of TTAGGG-hybridized DNA (Harley et al., 1990, Swanberg and Delany, 2003).

In order to measure percent telomeric DNA present in one sample relative to a calibrator sample (Harley et al., 1990), DNA is restricted, separated by gel electrophoresis, Southern blotted, and hybridized as with the determination of mean TRF length. However, rather than taking densitometry readings at discrete locations along the length of the smear of telomeric DNA, total telomeric DNA is measured by calculating the total integrated signal ODi over the same range of fragment sizes used for mean TRF analysis. Total integrated signal in this range is measured in each lane of any given gel, and results are expressed as a percentage of the signal from the earliest passage (Harley et al., 1990; Swanberg and Delany, 2003). The measurement of TRFs reveals a high degree of variability within cell lines prepared from single embryos of a highly inbred line, and mean TRF measurements are also subject to variability resulting from drift in the subpopulations within a culture. Therefore it is advisable to assay using more than one method to obtain a biologically relevant picture of telomere attrition or erosion (Swanberg and Delany, 2003).

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