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Two-Tailed Comet Assay (2T-Comet): Simultaneous Detection of DNA Single and Double Strand Breaks

  • Elva I. Cortés-GutiérrezEmail author
  • José Luis Fernández
  • Martha I. Dávila-Rodríguez
  • Carmen López-Fernández
  • Jaime Gosálvez
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1560)

Abstract

A modification of the original comet assay was developed for the simultaneous evaluation of DNA single strand breaks (SSBs) and double strand breaks (DSBs) in human spermatozoa. The two-dimensional perpendicular tail comet assay (2T-comet) combines non-denaturing and denaturant conditions to the same sperm nucleoid. In this case, the species-specific deproteinized sperm is first subjected to an electrophoretic field under non-denaturing conditions to mobilize isolated free discrete DNA fragments produced from DSBs; this is then followed by a second electrophoresis running perpendicular to the first one but under alkaline conditions to produce DNA denaturation, exposing SSBs on the same linear DNA chain or DNA fragments flanked by DSBs. This procedure results in a two dimensional comet tail emerging from the core where two types of original DNA affected molecule can be simultaneously discriminated. The 2T-comet is a fast, sensitive, and reliable procedure to distinguish between single and double strand DNA damage within the same cell. It is an innovative method for assessing sperm DNA integrity, which has important implications for human fertility and andrological pathology. This technique may be adapted to assess different DNA break types in other species and other cell types.

Key words

Two-tailed comet assay (2T-Comet) DNA fragmentation Single cell gel electrophoresis (SCGE) Single-stranded DNA breaks (SSB) Double-stranded DNA breaks (DSB) Spermatozoa 

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Elva I. Cortés-Gutiérrez
    • 1
    Email author
  • José Luis Fernández
    • 2
  • Martha I. Dávila-Rodríguez
    • 1
  • Carmen López-Fernández
    • 3
  • Jaime Gosálvez
    • 3
  1. 1.Department of Genetics, Centro de Investigación Biomédica del NoresteInstituto Mexicano del Seguro SocialMonterreyMexico
  2. 2.Genetics Unit, INIBIC, Complejo Hospitalario Universitario A Coruña and Laboratorio de Genética Molecular y RadiobiologíaCentro Oncológico de GaliciaLa CoruñaSpain
  3. 3.Unit of Genetics, Department of BiologyUniversidad Autónoma de MadridMadridSpain

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