Clipping of Telomere from Human Chromosomes Using a Chemistry‐Based Artificial Restriction DNA Cutter

Takumi Ishizuka1, Yan Xu1, Makoto Komiyama2

1 Division of Chemistry, Department of Medical Sciences, University of Miyazaki, Miyazaki, 2 Life Science Center of Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 6.13
DOI:  10.1002/0471142700.nc0613s61
Online Posting Date:  June, 2015
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Abstract

The detection of individual telomere lengths of human chromosomes can provide crucial information on genome stability, cancer, and telomere‐related diseases. However, current methods to measure telomere length entail shortcomings that have limited their use. Recently, we have developed a method for detection of individual telomere lengths (DITL) that uses a chemistry‐based DNA‐cutting approach. The most beneficial feature of the DITL approach is to cleave the sequence adjacent to the telomere followed by resolution of the telomere length at the nucleotide level of a single chromosome. In this unit, a protocol for successful detection of individual telomere lengths from individual chromosomes is described in detail. © 2015 by John Wiley & Sons, Inc.

Keywords: telomere length; DNA cleavage; peptide nucleic acid; biotechnology

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Detection and Measurement of Individual Telomere Lengths at a Single Chromosome Terminus by a Chemistry‐Based Method
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection and Measurement of Individual Telomere Lengths at a Single Chromosome Terminus by a Chemistry‐Based Method

  Materials
  • Pseudo‐complementary peptide nucleic acids (pcPNAs)
  • 3,5‐dimethoxy‐4‐hydroxycinnamic acid (Aldrich, cat. no. 85429), matrix for MALDI‐TOF mass spectrometry
  • Acetonitrile (Aldrich, cat. no. 34967)
  • Trifluoroacetic acid (TFA, Aldrich, cat. no. 302031)
  • Ce(NH 4) 2(NO 3) 6 (Tokyo Chemical Industry, cat. no. C1806)
  • 0.5 M EDTA, pH 8.0 (see appendix 2A)
  • 50 mM HEPES buffer, pH 7.0 (HEPES, Tokyo Chemical Industry, cat. no. H0396)
  • 1 M NaOH (see appendix 2A)
  • 7 × 106 cultured human cells, HEK 293 or HeLa
  • Blood & Cell Culture DNA Maxi Kit (QIAGEN, cat. no. 13362), to prepare whole human genomic DNA
  • 1 M NaCl solution (from 5 M stock; see appendix 2A)
  • 10 mM EDTPO solution, pH 7.0 (N,N,N',N'‐Ethylenediaminetetrakis(methylenephosphonic acid), Tokyo Chemical Industry, cat. no. E0393), to stop the scission reaction
  • TeloTAGGG Telomere Length Assay (Roche, cat. no. 12209136001), includes DIG molecular weight markers, DIG Easy Hyb buffer, DIG‐labeled telomere probe, and 10× blocking solution
  • Pulsed‐field‐certified agarose (BIO‐RAD, cat. no. 162‐0137)
  • 0.5× TBE buffer (from 10× TBE buffer stock; SIGMA, cat. no. T4415; see also appendix 2A)
  • TeloTAGGG Telomere Length Assay (Roche, cat. no. 12209136001)
  • Depurination solution (0.25 M HCl)
  • Denaturation solution (see recipe)
  • Neutralization solution (see recipe)
  • SSC Buffer, 20× concentrate (SIGMA, cat. no. S6639)
  • Stringent wash buffer I (see recipe)
  • Stringent wash buffer II (see recipe)
  • Washing buffer (see recipe)
  • Maleic acid buffer (see recipe)
  • Detection buffer (see recipe)
  • Anti‐Digoxigenin‐AP, Fab fragments (150 U, 200 μL, Roche, cat. no. 11093274910)
  • CDP‐Star, ready‐to‐use (Roche, cat. no. 12041677001)
  • 1.5‐mL microcentrifuge tubes
  • MALDI‐TOF mass spectrometer (Bruker Autoflex)
  • Nanodrop™ spectrophotometer (Thermo Scientific)
  • Isothermal bath (e.g., water bath or heat block)
  • Centrifugal evaporator (TOMY)
  • illustra MicroSpin S‐300 HR columns (GE Healthcare, cat. no. 27‐5130‐01)
  • CHEF Mapper™ pulsed‐field gel electrophoresis system (BIO‐RAD) equipped with cooling module (cat. no. 170‐3688), variable‐speed pump (cat. no. 170‐3644), standard casting stand (cat. no. 170‐3689)
  • Southern blot equipment
  • Nylon membranes, positively charged (Roche, cat. no. 11209272001)
  • UV‐crosslinker or Transilluminator
  • Hybridization bags (Roche, cat. no. 11666649001)
  • Orbital shaker
  • Heat sealer
  • Microcentrifuge (TOMY)
  • Biomolecular imager (e.g., LAS 4000, GE Healthcare) with analysis software (e.g., ImageQuant™, GE Healthcare)
  • Additional reagents and equipment for synthesis and purification of peptide nucleic acids (unit 4.11; Braasch et al., ), MALDI‐TOF mass spectrometry of oligonucleotides (unit 10.1; Castleberry et al., ), and Southern blotting (Brown, )
NOTE: Use nuclease‐free distilled water in all recipes and protocol steps.
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Figures

Videos

Literature Cited

Literature Cited
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