Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths

Abstract

In this protocol we describe a method to obtain telomere length parameters using Southern blots of terminal restriction fragments (TRFs). We use this approach primarily for epidemiological studies that examine leukocyte telomere length. However, the method can be adapted for telomere length measurements in other cells whose telomere lengths are within its detection boundaries. After extraction, DNA is inspected for integrity, digested, resolved by gel electrophoresis, transferred to a membrane, hybridized with labeled probes and exposed to X-ray film using chemiluminescence. Although precise and highly accurate, the method requires a considerable amount of DNA (3 μg per sample) and it measures both the canonical and noncanonical components of telomeres. The method also provides parameters of telomere length distribution in each DNA sample, which are useful in answering questions beyond those focusing on the mean length of telomeres in a given sample. A skilled technician can measure TRF length in 130 samples per week.

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Figure 1
Figure 2: Evaluation of DNA integrity.
Figure 3: Restriction digestion of genomic DNA.
Figure 4: Resolution of TRF length analysis in agarose gels of various percentages.
Figure 5: Representative layout of a 40-well gel.
Figure 6: Vacuum blotter setup for DNA transfer.
Figure 7: Distribution of TRF lengths in a representative sample.
Figure 8: Southern blot analysis of TRFs in leukocytes.
Figure 9: Appearance of a TRF signal suggesting DNA degradation before enzymatic digestion.

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Acknowledgements

Telomere research at the Center for Human Development and Aging, University of Medicine and Dentistry of New Jersey, New Jersey Medical School was supported by the National Institute of Aging (NIH grants R01AG021593, R01AG20132, R01AG030678 and 5F30AG032858).

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Contributions

M.K. oversaw the development and refinement of the TRF method, generated data shown in the figures and contributed to the writing of the article. R.S. assembled the elements of the paper, focusing on the presentation and writing of the experimental design and methodology. S.H. participated in the writing of the article, focusing on the statistical component of the introduction. J.S. oversaw the modeling and curve-fitting statistics for the TRF length distribution and contributed to the writing of the article. X.L. and X.C. routinely measured TRF length in our laboratory and provided R.S. with information to construct detailed protocols. C.H., who was a central figure in the development of the quantitative method for measuring the TRF distribution, contributed to the writing of the article. A.A. oversaw all elements of the project and participated in the writing of the article.

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Correspondence to Masayuki Kimura.

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Competing interests

C.B.H. holds stock in and consults for Geron Corporation, and is involved in the commercialization of telomere length assays.

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Kimura, M., Stone, R., Hunt, S. et al. Measurement of telomere length by the Southern blot analysis of terminal restriction fragment lengths. Nat Protoc 5, 1596–1607 (2010). https://doi.org/10.1038/nprot.2010.124

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