Amplification of padlock probes for DNA diagnostics by cascade rolling circle amplification or the polymerase chain reaction.
Amplification of padlock probes for DNA diagnostics by cascade rolling circle amplification or the polymerase chain reaction.
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DOI：
10.1043/1543-2165-123.20.1170
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发表时间：
2009-10
影响因子：
4.6
通讯作者：
David C. Thomas;G. Nardone;S. Randall
中科院分区：
文献类型：
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作者：
David C. Thomas;G. Nardone;S. Randall
挂锁探针是用于DNA诊断的高度特异性试剂，其可以区分具有单碱基突变的基因序列。当寡核苷酸探针的3'和5'末端区域在靶DNA序列上并置时，它们可以通过酶促连接环化并变得拓扑锁定于靶。然而，为了在基于溶液的诊断中有用，挂锁探针的灵敏度必须显著增强。目的描述两种环化锁式探针的几何扩增方法。设计Cascade rolling circle amplification是一种等温系统，使用通用引物和具有强链置换活性的DNA聚合酶，通过结合滚环复制和链置换合成的机制来扩增环化探针。其中一个引物被设计为能量转移标记的引物，其仅在掺入扩增产物中时产生荧光信号，从而实现直接检测手段。结果以pUC 19为模型靶环化89个碱基的探针，用Bst DNA聚合酶（大片段）在1h内从10个探针分子开始扩增100亿倍。聚合酶链反应也被用来扩增连接的挂锁探针在一个罕见的目标检测系统。在含有正常和突变型p53或c-Ki-ras 2基因靶序列的混合实验中，在存在500倍过量的正常靶拷贝的情况下容易检测到突变型靶。结论锁式探针可以扩增到溶液DNA诊断所需的高水平。
CONTEXT Padlock probes are highly specific reagents for DNA diagnostics that can discriminate gene sequences with single base mutations. When the 3' and 5' terminal regions of the oligonucleotide probes are juxtaposed on a target DNA sequence, they can be circularized by enzymatic ligation and become topologically locked to the target. However, to be useful in solution-based diagnostics, the sensitivity of padlock probes must be markedly enhanced. OBJECTIVE To describe two methods for geometric amplification of circularized padlock probes. DESIGN Cascade rolling circle amplification is an isothermal system that uses generic primers and a DNA polymerase with strong strand displacement activity to amplify circularized probes by a mechanism combining rolling circle replication and strand displacement synthesis. One of the primers was designed as an energy transfer-labeled primer, which generates a fluorescence signal only when incorporated into the amplified product, enabling a direct means of detection. RESULTS Using pUC19 as a model target to circularize an 89-base probe, a 10 billion-fold amplification was achieved with Bst DNA polymerase (large fragment) within 1 hour starting with as few as 10 probe molecules. The polymerase chain reaction was also used to amplify ligated padlock probes in a rare target detection system. In mixing experiments containing both normal and mutant p53 or c-Ki-ras2 gene target sequences, mutant targets were easily detected in the presence of a 500-fold excess of normal target copies. CONCLUSION These results indicate that padlock probes can be amplified to the high levels required for solution-based DNA diagnostics.