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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:& N4 q5 K+ \0 U- E( J) u; U
The Comet-FISH technique is a useful tool to detect overall and region-specific DNA damage and repair in individual cells. It combines two
6 l* `# [1 E" `: V4 g) R1 Bwell-established methods, the Comet assay (single cell gel electrophoresis) and the technique of fluorescence in situ hybridization (FISH). Whereas
( i$ Y7 G( ]+ `the Comet assay allows separating fragmented from non-fragmented DNA, FISH helps to detect specifically labelled DNA sequences of interest,0 v# t$ R0 B' [) `) }1 |# Q
including whole chromosomes. Thus the combination of both techniques has been applied in particular for detection of site-specific breaks in DNA
z0 x }( k# Q# u! i- eregions which are relevant for development of different diseases. This paper reviews the relevant literature and presents three examples on how
4 K4 o1 i$ O: yComet-FISH was used for studying the induction of DNA damage by genotoxic compounds related to oxidative stress in colon cancer-relevant
+ @' B2 `, |, f5 g6 r% I* D4 Ngenes (TP53, APC, KRAS) of a colon adenoma cell line. The accumulated evidence on relative sensitivity of these genes in comparison to global
5 b* ?9 y, T- u8 n1 i; idamage allows a more definite conclusion on the possible contribution of the genotoxic factors during colorectal carcinogenesis. Telomere fragility
, L2 F! ^ D$ E0 C3 nwas compared in different cell lines treated with cytostatic agents, and revealed new patterns of biological activities through the drugs and different) g7 W' i/ g6 i* O7 I
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
" {( T6 M; q$ W* K4 j# tregions using Comet-FISH, a method that can be developed to biomarker application. Taken together, available data suggests that Comet-FISH
7 G' I$ h4 `) [# `helps to get further insights into sensitivity of specific DNA regions and consequently in mechanisms of carcinogenesis. Although the nature of the
8 G+ _- B1 @- U/ Rmeasured Comet-FISH endpoint precludes us from stating basically that damage and repair are occurring within the specific gene, it is at least/ r7 A7 j+ M" ^2 M
possible to evaluate whether the damage and repair are occurring within the vicinity of the gene of interest. Q1 X2 j' J6 B5 f
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发表于 2012-3-26 10:54
