Mazzucco Giulia, Huda Armela, Galli Martina, Zanella Elia, Doksani Ylli
IFOM, FIRC Institute of Molecular Oncology, Milan, Italy.
Nat Protoc. 2022 Jun;17(6):1444-1467. doi: 10.1038/s41596-022-00684-9. Epub 2022 Apr 8.
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.
在此，我们提供了一种从小鼠和人类细胞中富集端粒重复序列的详细方案。该程序包括两轮连续的常用限制性内切酶消化，随后进行大小分级分离。大约需要2毫克基因组DNA，该程序持续5 - 6天，端粒富集倍数>800倍。纯化后的材料适用于端粒结构的单分子分析、通过电子显微镜观察端粒复制和重组中间体或在端粒重复序列上进行分子梳分析。富集程序不需要特殊技能，不过在程序开始时及时收获大量平板需要一些协助。还提供了该方案的小规模版本，涉及一轮消化和纯化，需要200微克DNA，4天内端粒富集约50倍。后者可与特定标记相结合，用于复制DNA的单分子分析或端粒重复序列的长读长测序分析。基于使用不切割目标重复序列的限制性内切酶，这里描述的程序可适用于其他重复元件的富集。
