The assay developed uses the enzyme -exonuclease to enzymatically increase the signal obtained from a specifically designed labeled DNA probe. The basis of the approach was to use a probe sequence which had been designed to hybridize very specifically to a target DNA sequence. The unique feature of the probe is that it will contain a 5’ phosphate group. This 5’ phosphate group is essential for the recognition of -exonuclease, an enzyme which will degrade a strand of DNA in a duplex, as long as there is a 5’ phosphate group present. This new approach required modification of the probe in such a way that once it is degraded by the -exonuclease a signal will be produced. The beauty of this approach is the fact that the target is not digested during the assay, this means it remains intact to hybridise to another probe hence each target can theoretically interact with many probe sequences thus resulting in an increase in the amount of signal that can be detected per sequence, signal amplification occurs.
The initial approach utilised fluorescence detection since existing labelling methodologies could be used. Several different approaches were developed each based around the use of a fluorophore and quencher system, whereby when the probe was intact the fluorescence was quenched and after hybridisation to the target and subsequence digestion by the -exonuclease, the fluorophore and the quencher were separated and a fluorescence signal was observed. These assays proved to be highly successful and the only issue with taking these assays forward was that the signal amplification was difficult to prove using the fluorescence instrumentation available.
The fluorescence assay allowed the action of the enzyme to be studied and the conditions for the enzyme to be optimised. Unfortunately the enzyme was found to have significant single stranded activity i.e. the -exonuclease would also digest a proportion of single stranded probe with a 5’ phosphate when it was single stranded and not hybridised to the target. Therefore the fluorescence assay was used to optimise conditions in terms of buffer, pH, time, temperature etc to try to minimise the digestion of ssDNA and optimise the digestion of dsDNA. To further study this area a DTG funded student was aligned with the project to further study the action and specificity of the enzyme as well as further the assay work started during this funded project.
The development of assays which utilised SERS as the final optical detection methodology were developed alongside the fluorescence based assays. The synthesis of a specific probe which is designed to not give a SERS signal until it is digested by the specific enzyme proved to be challenging and is still ongoing. However, another SERS assay format was developed which utilised a separation step to remove any unhybridised SERS active probe which had the added advantage of overcoming the issues with digestion of ssDNA that was identified in the fluorescence based assays. This format was highly successful and not only allowed the detection of exact complement target DNA but was also successfully detected longer length PCR product targets.