Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA.
Conditionally fluorescent molecular probes for detecting single base changes in double-stranded DNA.
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核酸序列的微小变异可产生深远的表型后果。因此，可靠地区分密切相关的序列对于研究和临床应用非常重要。在这里，我们证明了条件荧光DNA探针能够区分目标DNA中单个碱基的变化。这些探针使用一种新的可编程机制，其中每个单核苷酸多态性产生两个热力学不稳定的错配气泡，而不是在典型的基于杂交的分析中形成的单一错配。要产生与预期目标相同的荧光，需要超过含有单个核苷酸多态性的目标的12,000倍，并且在广泛的条件下可靠地进行检测。利用这些探针，我们在大肠杆菌rpoB基因的198个碱基对子序列中检测到点突变。我们的探针由多个寡核苷酸片段构建，规避了合成限制，使长连续DNA序列能够被探测。
Small variations in nucleic acid sequences can have far-reaching phenotypic consequences. Reliably distinguishing closely related sequences is therefore important for research and clinical applications. Here, we demonstrate that conditionally fluorescent DNA probes are capable of distinguishing variations of a single base in a stretch of target DNA. These probes use a novel programmable mechanism in which each single nucleotide polymorphism generates two thermodynamically destabilizing mismatch bubbles rather than the single mismatch formed during typical hybridization-based assays. Up to 12,000-fold excess of a target containing a single nucleotide polymorphism is required to generate the same fluorescence as one equivalent of the intended target, and detection works reliably over a wide range of conditions. Using these probes we detected point mutations in a 198 base pair subsequence of the E. Coli rpoB gene. Our probes are constructed from multiple oligonucleotide fragments, circumventing synthesis limitations and enabling long continuous DNA sequences to be probed.
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