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Purification of mammalian telomeric DNA for single-molecule analysis

Abstract

Here we provide a detailed protocol for the enrichment of telomeric repeats from mouse and human cells. The procedure consists of two successive rounds of digestion with frequently cutting restriction enzymes followed by size fractionation. Around 2 mg of genomic DNA is required, and the procedure lasts 5–6 d and yields preparations enriched >800-fold in telomeres. The purified material is suitable for single-molecule analysis of telomere structure, visualizing telomere replication and recombination intermediates by electron microscopy or performing molecular combing at telomeric repeats. No special skills are required for the enrichment procedure, while some assistance is needed in harvesting a large number of plates in a timely fashion at the beginning of the procedure. A smaller-scale version of the protocol that involves one round of digestion and purification requires 200 µg of DNA and enriches telomeres ~50-fold in 4 d is also provided. The latter can be combined with specific labeling for single-molecule analysis of replicating DNA or for long-read sequencing analysis of telomeric repeats. The procedure described here can be adapted to the enrichment of other repetitive elements, based on the use of restriction enzymes that do not cut into the repeat of interest.

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Fig. 1: Size distribution of the extracted gDNA.
Fig. 2: Schematic representation of the steps of the telomere enrichment procedure.
Fig. 3: Example of the bulk DNA and telomere signal migration after the first digestion round with HinfI and MspI.
Fig. 4: Separation of the digested DNA on a sucrose gradient and enrichment of telomeric repeats.
Fig. 5: Second digestion round of the large-scale procedure.
Fig. 6: Recovery efficiency of a plasmid DNA prep (Addgene no. 24150) with the silica beads gel extraction kit.
Fig. 7: Quantification of telomere enrichment in dot-blot and single-molecule analysis of DNA fibers.
Fig. 8: A preparative agarose gel after the removal of the HMW area.

Data availability

All data generated or analyzed during this work are included in the published article. Source data are provided with this paper.

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Acknowledgements

We are grateful to D. Piccini, who assisted us in setting up the separation in sucrose gradient. We are grateful to the members of the IFOM Cell Biology Unit for their invaluable assistance with growing large cell cultures. We are grateful to the IFOM Imaging facility for assisting with the acquisition of single-molecule images. We thank C. Villa at IFOM for her assistance and expertise in the preparation of the Supplementary Video files. Y.D. lab is supported by the Associazione Italiana per la Ricerca sul Cancro, AIRC, IG 19901.

Author information

Authors and Affiliations

Authors

Contributions

A.H. contributed to the development of the procedure. M.G. performed the experiments of the small-scale enrichment procedure. E.Z. performed the large-scale enrichment procedure on the HT1080 cell line. G.M. performed the other experiments with the large-scale enrichment procedure and prepared the rest of the figures of the manuscript. Y.D. conceived the procedure, oversaw the development of the protocol and wrote the manuscript.

Corresponding author

Correspondence to Ylli Doksani.

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Competing interests

The authors declare no competing interests.

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Peer review information

Nature Protocols thanks Jack Griffith and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key reference using this protocol

Mazzucco, G. et al. Nat. Commun. 11, 5297 (2020): https://doi.org/10.1038/s41467-020-19139-4

Extended data

Extended Data Fig. 1 Single-molecule analysis of telomeric DNA enrichment.

Single-molecule analysis of telomere enrichment. SV40 MEFs DNA from a non-enriched sample, a sample enriched with the large-scale procedure and a sample enriched with the small-scale procedure were combed on a silanized glass coverslip with the procedure described in Box 2. IF: immunofluorescence, the DNA was denatured in situ and incubated first with an antibody against single-stranded DNA, which labels all DNA molecules (green) and subsequently with a fluorescent (TTAGGG)3 peptide nucleic acid (PNA) probe that labels the telomeric molecules (magenta).

Source data

Extended Data Fig. 2 Quantification of telomeric DNA enrichment from HT1080 cells.

Quantification of telomeric DNA enrichment from the human HT1080 cell line in a dot blot. After the large-scale procedure, the indicated amounts of DNA for each enrichment round were loaded on a dot blot and hybridized with a telomeric probe. Fold-enrichment numbers (from one experiment) indicate the telomeric signal per ng of DNA, expressed relative to the value of bulk DNA.

Source data

Extended Data Fig. 3 No DNA damage induced during fractionation in sucrose gradient and DNA concentration.

Control experiment showing that separation in sucrose gradient and concentration in Amicon ultra centrifugal filters does not induce detectable DNA damage. Equal amounts of a plasmid prep (Addgene no. 71116) were either stored at 4 °C (input) or separated in a sucrose gradient, collected and concentrated in Amicon ultra centrifugal filters. As a positive control, the same plasmid DNA was incubated with 0.3 µg ml−1 ethidium bromide in TE 1× and irradiated for 10 min with 254 nm UV in a stratalinker. Equal amounts of the three samples were loaded in the agarose gel shown. Note that, in the positive control, treated with UV, all supercoiled form of the plasmid is lost, while no loss of the supercoiled form is detected after separation in sucrose gradient and sample concentration.

Source data

Supplementary information

Supplementary Information

Supplementary Methods

Supplementary Video 1

Video showing the psoralen cross-linking procedure

Supplementary Video 2

Video showing the manual preparation of sucrose gradients

Supplementary Video 3

Video showing sample loading on the sucrose gradient

Supplementary Video 4

Video showing the collection of sucrose fractions

Source data

Source Data Fig. 1

Whole gels and membranes used in Fig. 1

Source Data Fig. 3

Whole gels and membranes used in Fig. 3

Source Data Fig. 4

Whole gels and membranes used in Fig. 4

Source Data Fig. 5

Whole gels and membranes used in Fig. 5

Source Data Fig. 6

Whole gel used in Fig. 6

Source Data Fig. 7

Whole membrane used in Fig. 7a

Source Data Fig. 7

Data used for the graph in Fig. 7b

Source Data Fig. 8

Whole gel used in Fig. 8

Source Data Extended Data Fig. 1

Images used in Extended Data Fig. 1

Source Data Extended Data Fig. 2

Whole membrane used in Extended Data Fig. 2

Source Data Extended Data Fig. 3

Whole gel used in Extended Data Fig. 3

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Mazzucco, G., Huda, A., Galli, M. et al. Purification of mammalian telomeric DNA for single-molecule analysis. Nat Protoc 17, 1444–1467 (2022). https://doi.org/10.1038/s41596-022-00684-9

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