ExoSERRS Amplification free direct genomic sequence analysis by optical spectroscopy

拟议的研究涉及检测与特定疾病状态相关的特定DNA序列，例如囊性纤维化或表明感染的存在，例如耐甲氧西林金黄色葡萄球菌（MRSA）感染。所提出的方法在速度和成本方面优于现有的检测方法，因为在检测样品中的特定序列之前不需要初始步骤来增加样品中的DNA量。这项研究使用了一种叫做表面增强共振拉曼散射（SERRS）的技术。如果特定波长的光被射向非常小的银金属片，即纳米颗粒，那么一些反射光就会改变波长。这种波长的变化与纳米颗粒表面的分子有关，并提供了可用于识别的分子指纹。金属被用来放大这种效应，可以用来研究单个分子。由于产生了分子的指纹光谱，因此混合物的组成可以很容易地识别而无需分离。如果被分析的分子具有发色团，即有色分子，则这些信号进一步增加。有色分子或标签可以附着在DNA上，使其能够产生强烈的SERRS信号。为了检测已定义的DNA序列，使用互补序列，该互补序列将特异性杂交并因此识别所需序列。既然添加了互补序列，它就可以被修改为包含一个标签，通常是一个只有在互补序列结合时才能“看到”的彩色分子。只有当目标DNA存在时，这个标签才会被看到，如果它不存在，互补序列就不会结合，也不会看到任何信号。互补序列可以以几种不同的方式设计，以便在信号与其补体结合时检测到信号。本文提出了一种新的方法，即将特殊的染料标签附着在DNA的互补片段上，这将不会给出SERRS信号。当这段DNA与目标DNA结合时，将添加一种酶来消化互补DNA，释放出特殊的染料标签，然后在溶液中释放出来，现在可以发出SERRS信号。只有当目标DNA存在时，信号才会出现。根据设想，探针的性质将允许另一个探针分子与目标结合，然后被破坏，释放更多的信号，等等。与目前依赖于目标的放大而不是信号的方法相比，这将允许在获得的信号中进行放大。这种酶只在特殊设计的探针与目标结合时起作用，当探针在溶液中以单链形式游离时，它不会消化探针并释放染料。这种方法的吸引力在于极高的灵敏度和多路复用能力的结合，即一次检测多个DNA目标的能力。最终目标是实现从临床相关样本中检测特定DNA序列的pcr无检测。

The research proposed involves the detection of specific DNA sequences which will relate to a particular disease state, such as cystic fibrosis or indicate the presence of an infection, such as Methicillin-resistant Staphylococcus aureus (MRSA) infection. The method proposed offers benefits over existing detection methods in terms of speed and cost since it would not require an initial step to increase the amount of DNA in the sample before detection of the specific sequence in the sample could be carried out.The research involves the use of a technique called surface enhanced resonance Raman scattering (SERRS). If light of a particular wavelength is directed onto very small pieces of silver metal, known as nanoparticles, then some of the reflected light will have changed wavelength. This change in wavelength is related to the molecules on the nanoparticles' surface and provides a molecular fingerprint that can be used for identification. The metal is used to amplify this effect and can be used to study a single molecule. Since a fingerprint spectrum of a molecule is produced, the composition of mixtures can easily be identified without separation. These signals are further increased if the molecule being analysed has a chromophore i.e. is a coloured molecule. Coloured molecules or labels can be attached to DNA giving it the ability to generate intense SERRS signals.To detect a defined DNA sequence a complementary sequence is used which will hybridise specifically and hence recognize the desired sequence. Since the complementary sequence is added it can be modified to incorporate a label, usually a coloured molecule which will only be 'seen' when the complementary sequence has bound. This label will only be seen when the target DNA is present, if it is not present the complementary sequence will not bind and no signal will be seen. The complementary sequence can be designed in several different ways to allow a signal to be detected when it binds to its complement. Here is proposed a new method whereby a special dye label is attached to the complementary piece of DNA which will not give a SERRS signal. When this piece of DNA binds to the target DNA, an enzyme will be added which will digest the complementary DNA, releasing the special dye label which will then be free in solution and now able to give a SERRS signal. The signal will only be present when the target DNA is present and it is envisaged that the nature of the probe will allow another probe molecule to then bind to the target and then be destroyed releasing more signal and so on. This will allow for an amplification in the signal obtained, compared to current methods which rely on amplification of the target rather than the signal. This enzyme only works when the specially designed probe is bound to the target and will not digest the probe and release the dye when it is free in solution and in a single stranded form. The attraction of this approach is the combination of extreme sensitivity and the multiplexing capacity i.e. the ability to detect multiple DNA targets at once. The ultimate aim is to achieve PCR-less detection of a specific DNA sequence from a clinically relevant sample.