A recent study, published in Clinical Chemistry, has presented a rapid nanopore genotyping strategy to enable amplification-free identification and classification of ctDNA mutations.
Detecting genetic variants
Our ability to rapidly and accurately identify genetic variants has garnered interest due to its broad medical diagnostic applications. For example, classification of antibiotic resistant pathogens, cancer diagnosis and controlling the spreading of pandemics.
Circulating tumour DNA (ctDNA) is a fragment of DNA shed into the bloodstream from tumours. These fragments harbour tumour-specific alterations and can be obtained from plasma, serum and other bodily fluids. The mutations identified from ctDNAs can be used as biomarkers for early detection of primary and recurrent cancer. Their identification can also affect treatment outcomes.
For widespread adoption, liquid biopsy tools must overcome the inherent low signal to noise ratio during the early stages of tumour development. Quantification of current ctDNA mutations rely on next generation sequencing. Nonetheless, these technologies struggle with providing sufficient sensitivity and multiplexing capabilities. Additionally, they involve lengthy sample preparation, including amplification steps that can introduce errors and they often require expensive reagents and instruments which limit their broad use.
Nanopore devices hold great potential for nucleic acid biomarker analysis. In particular, solid-state nanopores (ssNPs) which can be used to probe low concentrations of DNA molecules from a dilute solution. To-date the vast majority of nanopore sensing research has used non-clinical DNA molecules. The ability to efficiently target specific DNAs directly from clinical samples using nanopore sensors remains largely unknown.
Nanopore method
To enable multiplexed, specific and sensitive detection using ssNPs, researchers have developed an assay in which unique genetic variations are converted to a molecular form via sequence specific ligation. Specifically, researchers created fluorescently labelled short DNA reporter molecules. Colour conjugation with multiple fluorophores enabled a unique multi-colour signature for different mutations. Optical information was then obtained from the fluorescent signals emitted by the molecules during their passage through the pore.
In this proof-of-concept paper, the team utilised their method to detect the presence of low-quantity ERBB2 F310S and PIK3Ca H1047R breast cancer mutations. They detected these mutations from both plasmids and xenograft mice blood samples. They demonstrated that this method could distinguish between a wild type and mutated sample. The method was also able to differentiate between the different mutations within the same sample.
This method presents a rapid and low cost ctDNA analysis that completely circumvents PCR amplification and library preparation. The authors believe that this approach meets current unmet demands in terms of sensitivity, multiplexing and cost; thereby, opening new avenues for early diagnosis of cancer.
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