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. 2024 Oct 28;52(19):e96.
doi: 10.1093/nar/gkae812.

High-throughput single telomere analysis using DNA microarray and fluorescent in situ hybridization

Yun-Ling Zheng  1 , Xingjia Wu  1 , Madeline Williams  1 , Simon Verhulst  2 , Jue Lin  3 , Yusuke Takahashi  4 , Jian-Xing Ma  4 , Ying Wang  5
Affiliations

High-throughput single telomere analysis using DNA microarray and fluorescent in situ hybridization

Yun-Ling Zheng et al. Nucleic Acids Res. .

Abstract

The human telomere system is highly dynamic. Both short and long leucocyte average telomere lengths (aTL) are associated with an increased risk of cancer and early death, illustrating the complex relationship between TL and human health and the importance of assessing TL distributions with single TL analysis. A DNA microarray and telomere fluorescent in situ hybridization (DNA-array-FISH) approach was developed to measure the base-pair (bp) lengths of single telomeres. On average 32000 telomeres were measured per DNA sample with one microarray chip assaying 96 test DNA samples. Various telomere parameters, i.e. aTL and the frequency of short/long telomeres, were computed to delineate TL distribution. The intra-assay and inter-assay coefficient of variations of aTL ranged from 1.37% to 3.98%. The correlation coefficient (r) of aTL in repeated measurements ranged from 0.91 to 1.00, demonstrating high measurement precision. aTLs measured by DNA-array-FISH predicted aTLs measured by terminal restriction fragment (TRF) analysis with r ranging 0.87-0.99. A new accurate and high-throughput method has been developed to measure the bp lengths of single telomeres. The large number of single TL data provides an opportunity for an in-depth analysis of telomere dynamics and the complex relationship between telomere and age-related diseases.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
Schematic overview of the DNA-Array-FISH-based single telomere analysis workflow. (A) genomic DNA was digested with restriction enzymes and printed on amino-saline coated glass slides to make DNA microarray chips. (B) DNA chips were hybridized with a telomere specific PNA probe. (C) An array spot was scanned using a fluorescent microscopy image system under 60× oil objective. (D) Enlarged view of telomere signals. (E) A telomere standard curve. (F) Total fluorescent intensity of each telomere spot in the digitized images were quantified and converted into bp length using linear regression, based on the slope and intercept of the telomere standard curve. (G) TL distribution of a human genomic DNA.
Figure 2.
Figure 2.
Average telomere length distribution of 92 human blood samples. Telomere length was measured by DNA-array-FISH with HinfI & MnlI digested DNA.
Figure 3.
Figure 3.
Correlation of telomere parameters between HinfI & MnlI and hPhI & MnlI digested DNA of human blood samples (N = 24). (A) aTL in bp, r = 0.97; (B) telomere length variation (CV), r = 0.96; (C) telomere frequencies in 1–3 kb TL group, r = 0.97; (D) telomere frequencies in 10–20 kb TL group, r = 0.97.
Figure 4.
Figure 4.
Correlation of aTL (bp) between repeated measurements (r = 1.00). DNA samples were extracted from cultured UMUC3TERC cell lines harvested at seven passages (p0–p6) and were digested with HinfI & MnlI.
Figure 5.
Figure 5.
Correlation of aTL (bp) between repeated measurements by DNA-array-FISH of 92 human blood DNA samples (r = 0.91). DNA samples were digested with HinfI & MnlI.
Figure 6.
Figure 6.
Correlation of aTL (bp) between TRF and DNA-array-FISH (r = 0.99). DNA samples were extracted from cultured UMUC3TERC cell lines harvested at seven passages (p0–p6) and were digested with HphI & MnlI (TRF) or HinfI & MnlI (DNA-array-FISH).
Figure 7.
Figure 7.
Correlation of aTL (bp) between TRF and DNA-array-FISH (r = 0.90). DNA samples (N = 27) were extracted from buffy coats and had been stored at –80°C for 12 years. DNA samples were digested with HinfI & RsaI (TRF) or HinfI & MnlI (DNA-array-FISH).
Figure 8.
Figure 8.
Average TL (bp) measured using DNA-array-FISH with AluI & HinfI digested DNA predicts aTL measured in the same samples using TRF with HinfI & RsaI digested DNA (r = 0.88).
Figure 9.
Figure 9.
Effect of age on telomere length (mean bp/year ± SE) at different percentiles of telomere length.
Figure 10.
Figure 10.
Telomere length distribution of a bladder cancer cell line, UMUC3, and a normal human blood DNA. DNA samples were digested with HinfI & MnlI.
Figure 11.
Figure 11.
Telomere length distribution of bladder cancer cell lines: UMUC3 and UMUC3TERC. UMUC3TERC were harvested at passages 1, 2, 3, 4, 5 and 6. DNA samples were digested with HinfI & MnlI.
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