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本帖最后由 细胞海洋 于 2012-3-26 13:34 编辑 & s& v; O" E4 h& X. F, X$ o
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Use of Comet-FISH in the study of DNA damage and repair:
3 F& G4 P% r3 Z) L8 v% \The Comet-FISH technique is a useful tool to detect overall and region-specific DNA damage and repair in individual cells. It combines two
4 [" p0 x& R7 G- ?( G8 zwell-established methods, the Comet assay (single cell gel electrophoresis) and the technique of fluorescence in situ hybridization (FISH). Whereas
1 f% \0 V6 _1 s9 C& Gthe Comet assay allows separating fragmented from non-fragmented DNA, FISH helps to detect specifically labelled DNA sequences of interest,
) a0 }2 B& Q# |9 rincluding whole chromosomes. Thus the combination of both techniques has been applied in particular for detection of site-specific breaks in DNA
v5 Q; I- T) n& Sregions which are relevant for development of different diseases. This paper reviews the relevant literature and presents three examples on how
o; _" q8 `* H6 H5 ?, [1 QComet-FISH was used for studying the induction of DNA damage by genotoxic compounds related to oxidative stress in colon cancer-relevant+ t- r) e7 {: X9 l$ n2 V
genes (TP53, APC, KRAS) of a colon adenoma cell line. The accumulated evidence on relative sensitivity of these genes in comparison to global. ~3 O/ u6 k3 m" x* N
damage allows a more definite conclusion on the possible contribution of the genotoxic factors during colorectal carcinogenesis. Telomere fragility
' t0 J x6 i8 S) o6 Q2 V9 @; {was compared in different cell lines treated with cytostatic agents, and revealed new patterns of biological activities through the drugs and different
( c, _* Z+ @+ e. asensitivities of the cell lines that were found to be associated with their tumour origin. A third example relates to measuring repair of specific gene
/ Y- G" z4 Y$ s: a- X# iregions using Comet-FISH, a method that can be developed to biomarker application. Taken together, available data suggests that Comet-FISH
# n2 Q3 f8 k" _4 x( P( n" d8 O, rhelps to get further insights into sensitivity of specific DNA regions and consequently in mechanisms of carcinogenesis. Although the nature of the
* E3 S4 C& c- K3 B& `, O" dmeasured Comet-FISH endpoint precludes us from stating basically that damage and repair are occurring within the specific gene, it is at least
3 a; C* f( M, l2 @* Bpossible to evaluate whether the damage and repair are occurring within the vicinity of the gene of interest.
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发表于 2012-3-26 10:54
