Novel Methods for Probing and Detecting Nucleic Acids

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Carter, Jake Giovanni ORCID: https://orcid.org/0000-0001-5623-6467 (2022). Novel Methods for Probing and Detecting Nucleic Acids. University of Birmingham. Ph.D.

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Abstract

Nucleic acids such as Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are essential for life in all organisms. As such the detection of nucleic acids can prove to be a useful tool in identifying the cause of an illness. The current method for detecting the presence of small quantities of nucleic acids is hinged on the polymerase chain reaction (PCR) the ‘gold standard’ of amplification, however, newer isothermal techniques that do not rely on temperature cycling such as loop mediated isothermal amplification (LAMP) are becoming more popular. However, the detection of the small quantities of nucleic acids found in pathogens still requires a lengthy process to increase the quantity of material prior to detection. Novel methods in which to amplify and subsequently detect nucleic acids were explored over the course of this research. Through the combination of a rapid isothermal amplification technique, and an established method that uses linear dichroism (LD) as a readout we demonstrated the ability to detect 10 fM of DNA in under 15 minutes. The detection of RNA requires a reverse transcription step to convert the RNA into DNA in order to be detected, we were able to develop an alternative method to detect the presence of viral RNA without the requirement of a length RT step. The results of this work demonstrate how the application of a reverse transcription free (RTF) step prior to an isothermal amplification technique, enables the detection of pathogenic material in less than 10 minutes. This offers an alternative, simpler, method for point of care (POC) testing. Work within the group has previously demonstrated the ability to detect DNA using M13 Bacteriophage based biosensors that are monitored using LD, unfortunately the ability to scale up production is limited; therefore, we investigated the ability of a synthetic foldamers to be measured using LD, offering a both a novel method for probing not only the structure of the foldamer itself, but also as a possible method for detecting nucleic acids.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):