A simple quenching method for fluorescence background reduction and its application to the direct, quantitative detection of specific mRNA
A simple quenching method for fluorescence background reduction and its application to the direct, quantitative detection of specific mRNA
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DOI：
10.1021/ac034803r
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发表时间：
2003-11-15
影响因子：
7.4
通讯作者：
Goodwin, PM
中科院分区：
文献类型：
--
作者：
Nolan, RL;Cai, H;Goodwin, PM
新的基因组序列信息正在迅速增加用于表征和治疗疾病的核酸(NA)靶标的数量。通过基于荧光的分析来检测这些目标通常受到来自大量存在于目标之上的未结合或未结合的探针的荧光背景的限制。为了解决这个问题，基于能量转移的探针已经被开发出来，并被用来减少来自非结合探针的荧光。尽管这些探头使NA目标探测发生了革命性的变化，但它们的使用需要严格注意设计限制、广泛的探头质量控制以及单独优化的实验条件。在这里，我们描述了一种更简单的背景降低方法，使用单标记猝灭低聚物来抑制探针-靶标杂交后多余的未结合探针荧光。大多数基于荧光的NA靶标检测和定量分析的第二个限制是需要对靶标或信号进行酶扩增以获得灵敏度。由于序列靶标和实验条件之间的扩增效率不同，扩增步骤使原始靶标拷贝数的量化成为问题。为了避免放大，我们将我们的猝灭方法与相关的双色单分子荧光检测相结合。我们展示了一个>；100倍的背景减少和检测的目标存在的浓度低至100fM使用双色分析。将这项技术应用于特定mRNA序列的检测和量化，使我们能够在不进行扩增步骤的情况下估计细胞来源的总RNA中的β-肌动蛋白拷贝数。
New genome sequence information is rapidly increasing the number of nucleic acid (NA) targets of use for characterizing and treating diseases. Detection of these targets by fluorescence-based assays is often limited by fluorescence background from unincorporated or unbound probes that are present in large excess over the target. To solve this problem, energy transfer-based probes have been developed and used to reduce the fluorescence from unbound probes. Although these probes have revolutionized NA target detection, their use requires scrupulous attention to design constraints, extensive probe quality control, and individually optimized experimental conditions. Here, we describe a simpler background reduction approach using singly labeled quencher oligomers to suppress excess unbound probe fluorescence following probe-target hybridization. A second limitation of most fluorescence-based NA target detection and quantification assays is the requirement for enzymatic amplification of target or signal for sensitivity. Amplification steps make quantification of original target copy number problematic because of variations in amplification efficiencies between the sequence targets and the experimental conditions. To avoid amplification, we coupled our quenching approach to a two-color NA assay with correlated, two-color, single-molecule fluorescence detection. We demonstrate a >100-fold background reduction and detection of targets present at concentrations as low as 100 fM using the two-color assay. The application of this technique to the detection and quantification of specific mRNA sequences enabled us to estimate beta-actin copy numbers in cell-derived total RNA without an amplification step.