Detection of nucleic acids

A method of detecting a difference in a nucleotide sequence of a first nucleic acid relative to a second nucleic acid, the method comprising:

• a) immobilizing a first single target nucleic acid molecule to a first discrete region of a solid support and immobilizing a second single target nucleic acid molecule to a second discrete region of said solid support, wherein the first target nucleic acid molecule comprises a first nucleotide sequence and the second target nucleic acid molecule comprises a second nucleotide sequence that is different than the first nucleotide sequence of the first target nucleic acid;
• b) providing detectably labeled query probes that associate and dissociate with one or both single target nucleic acids according to a kinetic rate constant kon or koff that is greater than 1 min−1;
• c) counting a plurality of time-resolved signal intensity transition events detected within said first discrete region and identifying the signal intensity transition events as a first candidate signal produced by the repeated association and dissociation of said detectably labeled query probes with said first single target nucleic acid molecule when the number of signal intensity transition events detected within the first discrete region is greater than a threshold value and counting a plurality of time-resolved signal intensity transition events detected within said second discrete region and identifying the signal intensity transition events as a second candidate signal produced by the repeated association and dissociation of said detectably labeled query probes with said second single target nucleic acid molecule when the number of signal intensity transition events detected, wherein said detectably labeled query probes all comprise the same known nucleotide sequence and the degree of complementarity between the detectably labeled query probes and the first target nucleic acid is different than the degree of complementarity between the detectably labeled query probes and the second target nucleic acid;
• d) determining from the first candidate signal a first value of a parameter characterizing query probe association and dissociation with said first single target nucleic acid molecule and determining from the second candidate signal a second value of said parameter characterizing query probe association and dissociation with said second single target nucleic acid molecule;
• and
• e) detecting a difference in the nucleotide sequence of the first nucleic acid relative to the second nucleic acid by comparing the first value of said parameter and the second value of said parameter, wherein a difference in the first and second values of said parameter indicates that the first and second target nucleic acids have different nucleotide sequences and wherein said parameter is selected from the group consisting of τon, τoff, mean τon, mean τoff, time-averaged τon, time-averaged τoff, number of transitions, mean number of transitions, distribution of the number of transitions, mean of the distribution of the number of transitions, median of the distribution of the number of transitions, peak of the distribution of the number of transitions, standard deviation of the distribution of the number of transitions, and shape of the distribution of the number of transitions.

A method for characterizing a target nucleic acid, the method comprising:

• a) immobilizing a single target nucleic acid molecule to a discrete region of a solid support and providing detectably labeled query probes that associate and dissociate with said single target nucleic acid according to a kinetic rate constant kon or koff that is greater than 1 min−1;
• b) counting a plurality of time-resolved signal intensity transition events detected within the discrete region and identifying the signal intensity transition events as a candidate signal produced by the repeated association and dissociation of said detectably labeled query probes with said immobilized single target nucleic acid molecule when the number of signal intensity transition events detected within the discrete region is greater than a threshold value;
• c) producing a kinetic fingerprint from the candidate signal;
• and
• d) using the kinetic fingerprint to characterize the immobilized target nucleic acid according to at least one of the following:
• 1) quantifying the nucleic acid in the sample;
• 2) determining the conformation of the nucleic acid;
• 3) determining the state of ligand binding to the target nucleic acid;
• or
• 4) quantifying a ligand bound to the target nucleic acid or quantifying a ligand not bound to the target nucleic acid.