Method

A method of diagnosing a disease in a subject, wherein the method comprises

• (i) contacting a sample comprising two or more target polynucleotides with a panel of two or more probes under conditions suitable for hybridisation of the target polynucleotides to the probes, wherein the target polynucleotides are markers of the disease, wherein the sample was obtained from the subject, and wherein:
• (a) each probe comprises a non-hybridisation region and a hybridisation region that specifically hybridises to one of the target polynucleotides to form a hybridised probe;
• (b) the hybridisation region of a probe of the panel comprises one or more non-natural nucleotides;
• and
• (c) none of the probes comprise a quadruplex-forming sequence:
• (ii) contacting the sample prepared in step (i) with a transmembrane pore through which a single stranded polynucleotide but not a double stranded polynucleotide can pass and applying a potential difference to the transmembrane pore such that the hybridised probes in the sample interact with the pore;
• (iii) measuring current blockades having a duration within a defined dwell time window, wherein:
• (a) the one or more non-natural nucleotides present in the hybridisation region of the probe increase or decrease the duration of the current blockade due to the probe hybridised to its target polynucleotide such that the proportion of current blockades that occur within the window due to the interaction of the hybridised probes with the pore is increased compared to when the corresponding one or more natural nucleotides are present in the hybridisation region;
• and
• (b) each hybridised probe gives rise to a current blockade indicative of that probe;
• and
• (iv) correlating the measured current blockades with the probes to determine the presence, absence or amount of the two or more target polynucleotides in the sample, thereby diagnosing the disease.