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本帖最后由 细胞海洋 于 2012-3-26 13:34 编辑
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Use of Comet-FISH in the study of DNA damage and repair:, N% {! o4 D2 B
The Comet-FISH technique is a useful tool to detect overall and region-specific DNA damage and repair in individual cells. It combines two2 y5 b: _ c& d. e% Z) p5 a
well-established methods, the Comet assay (single cell gel electrophoresis) and the technique of fluorescence in situ hybridization (FISH). Whereas
( E+ X3 L; w# V+ j8 Z" ^the Comet assay allows separating fragmented from non-fragmented DNA, FISH helps to detect specifically labelled DNA sequences of interest,
% a. e& h( q/ c. U! Dincluding whole chromosomes. Thus the combination of both techniques has been applied in particular for detection of site-specific breaks in DNA
( p$ c4 Q0 g, _6 _5 Oregions which are relevant for development of different diseases. This paper reviews the relevant literature and presents three examples on how2 \9 v8 S2 |6 r. P' D( }0 K
Comet-FISH was used for studying the induction of DNA damage by genotoxic compounds related to oxidative stress in colon cancer-relevant7 a5 e1 ~2 w' n' L& }
genes (TP53, APC, KRAS) of a colon adenoma cell line. The accumulated evidence on relative sensitivity of these genes in comparison to global
# r8 }, b }3 y0 j6 s( i+ O( fdamage allows a more definite conclusion on the possible contribution of the genotoxic factors during colorectal carcinogenesis. Telomere fragility* h% M1 j' z5 j/ N8 b$ v
was compared in different cell lines treated with cytostatic agents, and revealed new patterns of biological activities through the drugs and different5 F, h( a7 ^% T5 U
sensitivities of the cell lines that were found to be associated with their tumour origin. A third example relates to measuring repair of specific gene
, @9 Y6 i1 H2 sregions using Comet-FISH, a method that can be developed to biomarker application. Taken together, available data suggests that Comet-FISH
* i. s- T; f4 Z Zhelps to get further insights into sensitivity of specific DNA regions and consequently in mechanisms of carcinogenesis. Although the nature of the
9 Y$ e8 v3 p4 fmeasured Comet-FISH endpoint precludes us from stating basically that damage and repair are occurring within the specific gene, it is at least
( j: w' G$ l0 v$ R% M" N' ypossible 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
