Accurate DNA fragment sizing by capillary electrophoresis with laser-induced fluorescence array for detection of sequence specificity of DNA damage
Accurate DNA fragment sizing by capillary electrophoresis with laser-induced fluorescence array for detection of sequence specificity of DNA damage
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DOI：
10.1021/ac702265b
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发表时间：
2008-03-15
影响因子：
7.4
通讯作者：
Rusling, James F.
中科院分区：
文献类型：
--
作者：
Fundador, Erwin V.;Choudhary, Dharamainder;Rusling, James F.
癌症与编码关键生物分子的基因中特定密码子的突变有关，如肿瘤抑制蛋白(例如，p53)。激活的代谢物，如苯并[a]吡二醇环氧化物，作用于DNA的首选核苷酸序列，这种突变已在癌症中被发现。DNA反应部位的识别依赖于对受损部位链断裂产生的寡核苷酸片段大小的准确分析。在这里，我们报告了一种新的DNA片段大小的测定方法-毛细管电泳激光诱导荧光检测(CE-LIF)。采用CE-LIF双色荧光检测阵列，实现了绝对的施胶精度和速度。准确度取决于通过参照与样品片段具有相同序列和相同标签的初级标准品来校正商业标准品的结果。该方法是通过检测一个[...GGCGCGCAG...]环氧苯乙烷在代表细胞色素P1B1基因的寡核苷酸上的G反应位点。这种方法避免了对放射性同位素的需要，劳动强度较低，比P-32末端标记的替代PAGE更快。
Cancer has been linked to mutations within specific codons in genes that code for critical biomolecules such as tumor suppressor proteins (e.g., p53). Activated metabolites like benzo[a]pyrenediol epoxide act on preferred nucleotide sequences of DNA, and such mutations have been identified in cancers. DNA reaction site identification depends on accurate analysis of oligonucleotide fragment sizes produced by strand breakage at the damaged sites. Herein, we report a new method for DNA fragment sizing using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Absolute sizing accuracy and speed are achieved by utilizing a CE-LIF array with two-color fluorescence detection. Accuracy depends on correcting results with commercial standards by referring them to primary standards with the same sequences and identical labels as sample fragments. The method is demonstrated by detection of a [... GGCGCGCAG... ] G reaction site for styrene oxide on an oligonucleotide representing the CYP1B1 gene. This approach avoids the need for radioactive isotopes and is less labor intensive and faster than the alternative PAGE with P-32 end labeling.