Gene expression analysis using single molecule detection -: art. no. e89
Gene expression analysis using single molecule detection -: art. no. e89
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DOI：
10.1093/nar/gng089
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发表时间：
2003-08-15
影响因子：
14.9
通讯作者：
Rigler, R
中科院分区：
文献类型：
--
作者：
Korn, K;Gardellin, P;Rigler, R
单分子检测技术的最新发展，特别是荧光相关光谱（FCS）的引入，导致了生物研究中的一些重要应用。我们提出了一种独特的方法，用于基因表达分析使用双色互相关。表达测定基于两种染料标记的DNA探针与选定的靶基因的基因特异性杂交。溶液中双标记分子的计数允许以绝对数量定量表达的基因拷贝。由于FCS的检测和分析可以在单分子水平上进行，因此不需要任何类型的扩增。我们描述了基因表达测定和目前的数据表明这种新技术的能力。为了证明基因特异性，我们用基因缺失的总cDNA进行实验。通过定量从HL-60细胞制备的cDNA中选择的高、中、低丰度基因来证明生物学应用。
Recent developments of single molecule detection techniques and in particular the introduction of fluorescence correlation spectroscopy (FCS) led to a number of important applications in biological research. We present a unique approach for the gene expression analysis using dual-color cross-correlation. The expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target gene. The counting of the dual-labeled molecules within the solution allows the quantification of the expressed gene copies in absolute numbers. As detection and analysis by FCS can be performed at the level of single molecules, there is no need for any type of amplification. We describe the gene expression assay and present data demonstrating the capacity of this novel technology. In order to prove the gene specificity, we performed experiments with gene-depleted total cDNA. The biological application was demonstrated by quantifying selected high, medium and low abundant genes in cDNA prepared from HL-60 cells.