A method for determining the presence, absence or amount of two or more target polynucleotides in a sample comprising additional components, the method comprising: (i) contacting the sample with a panel of two or more probes under conditions suitable for hybridisation of the target polynucleotides to the probes, 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; and (b) the hybridisation region of a probe of the panel comprises one or more non-natural nucleotides; (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 probes in the sample move through the pore, wherein at least a portion of the non-hybridisation region of a probe in the sample enters the transmembrane pore and remains within the transmembrane pore until the target polynucleotide dehybridises from the hybridization region of the probe, and the probe then moves through the pore; (iii) measuring current blockades having a duration within a defined window that includes current blockades caused by probes bound to their respective targets and excludes longer and/or shorter current blockades, 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 compared to when the corresponding one or more natural nucleotides are present in the hybridisation region, such that the current blockades that occur due to the movement of the hybridised probes through the pore occur within the defined window; and (b) each hybridised probe gives rise to a current blockade signal pattern indicative of that probe; and (iv) correlating the measured current blockade signal patterns with the probes, thereby determining the presence, absence or amount of the two or more target polynucleotides in the sample. (i) contacting the sample with a panel of two or more probes under conditions suitable for hybridisation of the target polynucleotides to the probes, 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; and (b) the hybridisation region of a probe of the panel comprises one or more non-natural nucleotides; (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; and (b) the hybridisation region of a probe of the panel comprises one or more non-natural nucleotides; (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 probes in the sample move through the pore, wherein at least a portion of the non-hybridisation region of a probe in the sample enters the transmembrane pore and remains within the transmembrane pore until the target polynucleotide dehybridises from the hybridization region of the probe, and the probe then moves through the pore; (iii) measuring current blockades having a duration within a defined window that includes current blockades caused by probes bound to their respective targets and excludes longer and/or shorter current blockades, 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 compared to when the corresponding one or more natural nucleotides are present in the hybridisation region, such that the current blockades that occur due to the movement of the hybridised probes through the pore occur within the defined window; and (b) each hybridised probe gives rise to a current blockade signal pattern indicative of that probe; and (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 compared to when the corresponding one or more natural nucleotides are present in the hybridisation region, such that the current blockades that occur due to the movement of the hybridised probes through the pore occur within the defined window; and (b) each hybridised probe gives rise to a current blockade signal pattern indicative of that probe; and (iv) correlating the measured current blockade signal patterns with the probes, thereby determining the presence, absence or amount of the two or more target polynucleotides in the sample.