Laboratoire de Géologie de Lyon, Terre, Planètes, Environnement, UMR 5276, Université Lyon 1, CNRS, Ecole Normale Supérieure de Lyon, 46 allée d'Italie, 69364, Lyon Cedex 07, France.
Anal Bioanal Chem. 2012 Aug;404(2):415-22. doi: 10.1007/s00216-012-6133-1. Epub 2012 Jun 14.
We have developed a fully enzyme-free SERRS hybridization assay for specific detection of double-stranded DNA sequences. Although all DNA detection methods ranging from PCR to high-throughput sequencing rely on enzymes, this method is unique for being totally non-enzymatic. The efficiency of enzymatic processes is affected by alterations, modifications, and/or quality of DNA. For instance, a limitation of most DNA polymerases is their inability to process DNA damaged by blocking lesions. As a result, enzymatic amplification and sequencing of degraded DNA often fail. In this study we succeeded in detecting and quantifying, within a mixture, relative amounts of closely related double-stranded DNA sequences from Rupicapra rupicapra (chamois) and Capra hircus (goat). The non-enzymatic SERRS assay presented here is the corner stone of a promising approach to overcome the failure of DNA polymerase when DNA is too degraded or when the concentration of polymerase inhibitors is too high. It is the first time double-stranded DNA has been detected with a truly non-enzymatic SERRS-based method. This non-enzymatic, inexpensive, rapid assay is therefore a breakthrough in nucleic acid detection.
我们开发了一种完全无酶的 SERRS 杂交分析方法,用于特异性检测双链 DNA 序列。虽然从 PCR 到高通量测序的所有 DNA 检测方法都依赖于酶,但这种方法是独一无二的,因为它完全是非酶的。酶促过程的效率受到 DNA 的改变、修饰和/或质量的影响。例如,大多数 DNA 聚合酶的一个局限性是它们无法处理被阻断损伤的 DNA。因此,降解 DNA 的酶促扩增和测序经常失败。在这项研究中,我们成功地在混合物中检测和定量了来自岩羚羊(Rupicapra rupicapra)和山羊(Capra hircus)的密切相关双链 DNA 序列的相对含量。这里提出的非酶 SERRS 分析是一种很有前途的方法的基石,可以克服当 DNA 降解太严重或聚合酶抑制剂浓度过高时 DNA 聚合酶失效的问题。这是第一次使用真正的非酶 SERRS 方法检测双链 DNA。这种非酶、廉价、快速的检测方法是核酸检测的一个突破。