ExoSERRS Amplification free direct genomic sequence analysis by optical spectroscopy

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.