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

Cortés‐Gutiérrez, Elva I.; Fernández, José Luis Fernández; Dávila-Rodríguez, Martha I.; López‐Fernández, Carmen; Gosálvez, Jaime

doi:10.1007/978-1-4939-6788-9_22

Cited by 28 publications

(19 citation statements)

References 10 publications

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“…, either neutral (DSBs) or alkaline (SSBs and DSBs), this distinction can be made only by the Comet assay [ 120 ]. The two-tailed Comet assay can directly differentiate between the SSBs and DSBs [ 121 ]. On the other hand, the newest SDF test introduced for the immunodetection of gamma histone 2AX (γH2AX), can only assess DSBs [ 115 ].…”

Section: Sperm Dna Fragmentation: Single- Versus Dmentioning

confidence: 99%

Sperm DNA Fragmentation: A New Guideline for Clinicians

Agarwal

Majzoub

Baskaran

et al. 2020

World J Mens Health

187

144

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Sperm DNA integrity is crucial for fertilization and development of healthy offspring. The spermatozoon undergoes extensive molecular remodeling of its nucleus during later phases of spermatogenesis, which imparts compaction and protects the genetic content. Testicular (defective maturation and abortive apoptosis) and post-testicular (oxidative stress) mechanisms are implicated in the etiology of sperm DNA fragmentation (SDF), which affects both natural and assisted reproduction. Several clinical and environmental factors are known to negatively impact sperm DNA integrity. An increasing number of reports emphasizes the direct relationship between sperm DNA damage and male infertility. Currently, several assays are available to assess sperm DNA damage, however, routine assessment of SDF in clinical practice is not recommended by professional organizations. This article provides an overview of SDF types, origin and comparative analysis of various SDF assays while primarily focusing on the clinical indications of SDF testing. Importantly, we report four clinical cases where SDF testing had played a significant role in improving fertility outcome. In light of these clinical case reports and recent scientific evidence, this review provides expert recommendations on SDF testing and examines the advantages and drawbacks of the clinical utility of SDF testing using Strength-Weaknesses-Opportunities-Threats (SWOT) analysis.

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Section: Sperm Dna Fragmentation: Single- Versus Dmentioning

confidence: 99%

Sperm DNA Fragmentation: A New Guideline for Clinicians

Agarwal

Majzoub

Baskaran

et al. 2020

World J Mens Health

187

144

View full text Add to dashboard Cite

show abstract

“…Recently, this separation has been shown to occur also within a combination of denaturing and nondenaturing conditions. This approach enables simultaneous evaluation of ssDNA and dsDNA strand breaks and thus is called 2T-comet assay, short for "two-dimensional perpendicular tail comet assay" [78]. It has been used for detecting DNA strand breaks in human spermatozoa.…”

Section: Comet Assaymentioning

confidence: 99%

An overview of apoptosis assays detecting DNA fragmentation

2018

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Apoptosis has been recognized as a type of programmed cell death connected with characteristic morphological and biochemical changes in cells. This programmed cell death plays an important role in the genesis of a number of physiological and pathological processes. Thus, it can be very important to detect the signs of apoptosis in a study of cellular metabolism. The present paper provides an overview of methods often being used for detecting DNA fragmentation as one of the most specific findings in apoptosis. To date, three routine assays have been developed for detecting DNA fragmentation: DNA ladder assay, TUNEL assay, and comet assay. All these methods differ in their principles for detecting DNA fragmentation. DNA ladder assay detects the characteristic "DNA ladder" pattern formed during internucleosomal cleavage of DNA. Terminal deoxynUcleotidyl transferase Nick-End Labeling (TUNEL) assay detects DNA strand breaks using terminal deoxynucleotidyl transferase catalyzing attachment of modified deoxynucleotides on the DNA strand breaks. Comet assay can be used for detecting nucleus breakdown producing single/double-strand DNA breaks. The aim of this review is to describe the present knowledge on these three methods, including optimized approaches, techniques, and limitations.

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“…In general, it is assumed that the comet assay targets DSBs, but the alkaline comet assay can be used to detect and differentiate both DSBs and SSBs; using an extra step in the comet assay. It is possible to assess the simultaneous presence of DSBs and SSBs in a single cell using a two-dimensional or two-tailed comet assay [94].…”

Section: Dna Damage In Reproductive Cellsmentioning

confidence: 99%

DNA Damage and Repair in Human Reproductive Cells

García-Rodríguez

Gosálvez

Agarwal

et al. 2018

IJMS

Self Cite

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The fundamental underlying paradigm of sexual reproduction is the production of male and female gametes of sufficient genetic difference and quality that, following syngamy, they result in embryos with genomic potential to allow for future adaptive change and the ability to respond to selective pressure. The fusion of dissimilar gametes resulting in the formation of a normal and viable embryo is known as anisogamy, and is concomitant with precise structural, physiological, and molecular control of gamete function for species survival. However, along the reproductive life cycle of all organisms, both male and female gametes can be exposed to an array of “stressors” that may adversely affect the composition and biological integrity of their proteins, lipids and nucleic acids, that may consequently compromise their capacity to produce normal embryos. The aim of this review is to highlight gamete genome organization, differences in the chronology of gamete production between the male and female, the inherent DNA protective mechanisms in these reproductive cells, the aetiology of DNA damage in germ cells, and the remarkable DNA repair mechanisms, pre- and post-syngamy, that function to maintain genome integrity.

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

Cited by 28 publications

References 10 publications

Sperm DNA Fragmentation: A New Guideline for Clinicians

Sperm DNA Fragmentation: A New Guideline for Clinicians

An overview of apoptosis assays detecting DNA fragmentation

DNA Damage and Repair in Human Reproductive Cells

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