Alkaline Single Cell Gel Electrophoresis: A Sensitive Technique for Quantitative Estimation of DNA Damage in Individual Cancer Cells Following Chemotherapy

Published: April 30, 2023

Abstract

Source: Lu, Y., et al. Evaluating In Vitro DNA Damage Using Comet Assay J. Vis. Exp. (2017)

In this video, we demonstrate an alkaline single-cell electrophoresis assay to quantify both single- and double-strand DNA breaks in doxorubicin-treated cells. Alkaline conditions disrupt the hydrogen bonds between DNA base pairs, resulting in the complete unwinding and separation of DNA strands. This allows differential migration of undamaged and damaged DNA fragments in agarose gel upon electrophoresis, revealing a comet-shaped structure of slowly migrating undamaged DNA forming the circular comet head, and rapidly migrating damaged DNA fragments forming the elongated comet tail.

Protocol

1. Prepare Reagents 1x PBS Dilute 100 mL 10x PBS with 900 mL dH2O and adjust the pH to 7.4 using a pH meter. Store at room temperature. Lysis solution (LS) Prepare 2.5 M NaCl, 100 mM disodium EDTA, 10 mM Tris base, and 200 mM NaOH in 900 mL dH2O; it commonly takes about 20 min to allow the mixture to fully dissolve. Adjust the pH to 10 using a pH meter. Add 1% sodium lauryl sarcosinate and …

Divulgazioni

The authors have nothing to disclose.

Materials

10x PBS(Ca++, Mg++ free)  TEKnova  P0196
NaCl  Sigma  S5886
EDTA  TEKnova  E0308
Trizma base  Sigma  T1503
NaOH  Sigma  72068
Sodium lauryl sarcosinate  Sigma  L7414
Triton X-100  Sigma  93443
SYBR Green  Invitrogen  S33102
Low melting point agarose  Invitrogen  16520
Agarose  Invitrogen  16500
95% ethanol  WARNER-GRAHAM  #64-17-5
Trypsin  GIBCO  25300-054
Glass tissue slides  ELECTRON MICROSCOPY SCIENCES  63422-11
Kimwipes  KIMberly-Clark
1.5 mL Microcentrifuge Tubes  DENVILLE
Pipette Tips  SHARP
Microwave  Avanti
Waterbath  PRECISION
Horizontal electrophoresis chamber  TREVIGEN  Cometassay ES II
Power supply  Bio-Rad
Incubator  Quincy Lab  Model 12-140E
Fluorescent microscope  Zeiss  LSM700
Micropipettor  Eppendorf

Play Video

Citazione di questo articolo
Alkaline Single Cell Gel Electrophoresis: A Sensitive Technique for Quantitative Estimation of DNA Damage in Individual Cancer Cells Following Chemotherapy. J. Vis. Exp. (Pending Publication), e21114, doi: (2023).

View Video