Methods employing oligonucleotide-binding e-tag probes

A method of detecting the presence or absence of each of a plurality of polynucleotides in a sample, the method comprising the steps of:

• providing for each polynucleotide a primer specific for a first region of the polynucleotide and an electrophoretic probe specific for a second region of the polynucleotide, the electrophoretic probe having a capture ligand attached and an eTag reporter attached, such that the eTag reporter of each electrophoretic probe has an electrophoretic mobility different from that of the eTag reporters of every other electrophoretic probe so that eTag reporters from different electrophoretic probes form distinct peaks in an electropherogram upon electrophoretic separation;
• combining in a mixture a nuclease, the sample, the electrophoretic probes, and the primers under conditions that allow the primers and the electrophoretic probes to hybridize to their respective polynucleotides to form complexes, the nuclease recognizing such complexes and digesting the electrophoretic probes therein so that eTag reporters are released;
• excluding from the electropherogram undigested electrophoretic probes by treating the mixture with a capture agent that specifically binds the capture ligands of the electrophoretic probes;
• and
• electrophoretically separating and identifying the released eTag reporters to determine the presence or absence of each of the plurality of polynucleotides.