PMID- 28645380
OWN - NLM
STAT- MEDLINE
DCOM- 20180402
LR  - 20181022
IS  - 1557-7988 (Electronic)
IS  - 0076-6879 (Linking)
VI  - 591
DP  - 2017
TI  - Comet-FISH for Ultrasensitive Strand-Specific Detection of DNA Damage in Single 
      Cells.
PG  - 83-95
LID - S0076-6879(17)30121-0 [pii]
LID - 10.1016/bs.mie.2017.03.023 [doi]
AB  - The genome integrity of living organisms is constantly threatened by endogenous 
      cellular metabolic processes and environmental agents. To quantify these low, 
      physiologically relevant levels of DNA damage, a single-cell gel electrophoresis 
      (comet) combined with strand-specific fluorescence in situ hybridization 
      (FISH)-based approach has been developed. This approach enables the 
      quantification of low levels of specific DNA lesions in each strand of the 
      selected sequence at the single-molecule sensitivity, as well as in the genome 
      overall in single cells. In this method, the percentage of DNA in the comet tail 
      is used to quantify lesions in the genome overall. Lesions in the respective 
      strands of the designated sequence are analyzed using strand-specific FISH 
      probes. These probes targeting the 3' and 5' termini of the selected sequence are 
      conjugated with two distinct fluorophores. Following the comet-FISH assay, the 
      two termini of the designated sequence are visualized as two spots with different 
      colors, under a fluorescence microscope. Separated spots indicate a damage 
      strand, while adjacent or colocalized spots imply an intact strand. Any DNA 
      lesions or DNA modifications, which can be converted into strand breaks 
      enzymatically or chemically, can be quantified by this method. The comet-FISH 
      approach described here can be applied to the study of the molecular mechanisms 
      of various repair pathways, as well as in drug screening to develop inhibitors 
      for specific repair pathways.
CI  - (c) 2017 Elsevier Inc. All rights reserved.
FAU - Mondal, Manas
AU  - Mondal M
AD  - Biodesign Institute & School of Molecular Sciences, Arizona State University, 
      Tempe, AZ, United States.
FAU - Guo, Jia
AU  - Guo J
AD  - Biodesign Institute & School of Molecular Sciences, Arizona State University, 
      Tempe, AZ, United States. Electronic address: jiaguo@asu.edu.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20170503
PL  - United States
TA  - Methods Enzymol
JT  - Methods in enzymology
JID - 0212271
RN  - 0 (Fluorescent Dyes)
SB  - IM
MH  - Animals
MH  - Comet Assay/*methods
MH  - *DNA Damage
MH  - DNA Repair
MH  - Fluorescent Dyes/chemistry
MH  - Humans
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Limit of Detection
MH  - *Single-Cell Analysis
OTO - NOTNLM
OT  - Fluorescence in situ hybridization
OT  - Oxidative DNA damage
OT  - Single cell gel electrophoresis
OT  - Transcription-coupled repair
EDAT- 2017/06/25 06:00
MHDA- 2018/04/03 06:00
CRDT- 2017/06/25 06:00
PHST- 2017/06/25 06:00 [entrez]
PHST- 2017/06/25 06:00 [pubmed]
PHST- 2018/04/03 06:00 [medline]
AID - S0076-6879(17)30121-0 [pii]
AID - 10.1016/bs.mie.2017.03.023 [doi]
PST - ppublish
SO  - Methods Enzymol. 2017;591:83-95. doi: 10.1016/bs.mie.2017.03.023. Epub 2017 May 
      3.