Gentica Molecular A

Comet Assay
Single Cell Gel Electrophoresis

Trabalho realizado por:

Andreia Medeiros n31871
Figura 1- In Increasing the resolution of the comet assay LBCM Turno Prtico P2
using fluorescent in situ hybridization[1]
Utilidade deste mtodo e suas vantagens:
Detectar danos presentes no DNA ao nvel de uma clula eucaritica
individual
Necessidade de poucas clulas por amostra
Baixo custo em relaco a outros mtodos
Fcil aplicao
Reduzido dispndio de tempo

Aplicaes:
Biomonitorizao
Testes de genotoxicidade
Nutrio
Reproduo assistida
 Procedimento experimental

1. Compartimentalizao das
clulas da suspenso em
agarose
2. Lise celular em condies
neutras ou alcalinas
3. Electroforese
4. Colorao
5. Anlise dos resultados

Figura 2- In DNA strand breakage in aquatic organisms and the potential

value of the comet assay in environmental monitoring [2]
 Anlise de resultados: o grau de danos no DNA
proporcional ao tamanho da cauda do cometa

A - Baixo grau de
danos no DNA

B e C - Grau
intermdio de danos
no DNA

D - Elevado grau de
danos no DNA

Figura 3- In The Relationship between Environmental Exposures to Phthalates and DNA Damage in Human
Sperm Using the Neutral Comet Assay [3]
Variantes tcnica
Comet Assay Alcalino
Comet Assay Neutro
FISH Comet Assay (localizar
cromossomas ou regies destes)
Comet assay com enzimas de
reparao do DNA (mais especfico)
Marcao com BUdR (marcao
durante a replicao e deteco com
o anticorpo)

Figura 4-In Increasing the resolution of the comet assay using

fluorescent in situ hybridization [1]
Bibliografia
[1] Shaposhnikov S, Frengen E, Collins AR (2009). Increasing the resolution of the comet assay
using fluorescent in situ hybridization--a review. Mutagenesis. 24(5):383-9.
[2] C.L. Mitchelmore and J.K. Chipman(November 1997). DNA strand breakage in aquatic
organisms and the potential value of the comet assay in environmental monitoring. Mutation
Research 399, 135147.
[3] Susan M. Duty, Narendra P. Singh, Manori J. Silva, Dana B. Barr, John W. Brock, Louise
Ryan, Robert F. Herrick, David C. Christiani and Russ Hauser (July 2003). The Relationship
between Environmental Exposures to Phthalates and DNA Damage in Human Sperm Using
the Neutral Comet Assay. Environmental Health Perspectives. 111(9).
[4] Maria Dusinska and Andrew R.Collins (March 2008). The comet assay in human
biomonitoring: geneenvironment interactions. Mutagenesis 23(3):191205.
[5] Graciela Spivak, Rachel A. Cox, and Philip C. Hanawalt (2007). New Applications of the
Comet Assay: Comet-FISH and Transcription-Coupled DNA Repair. Mutat Res. 681(1): 4450.
[6] Flvio Azevedo, Filipe Marques, Hanna Fokt, Rui Oliveira and Bjorn Johansson (September
2010). Measuring oxidative DNA damage and DNA repair using the yeast comet assay. Yeast
2011; 28: 5561.
[7] Peggy L Olive and Judit P Banth (June 2006). The comet assay: a method to measure DNA
damage in individual cells. Nature Protocols 1(1).