Correcting for false positive signals from mismatched probe-target binding in a multiplexed hybridization-mediated assay
Described are methods of assay design and assay image correction, useful for multiplexed genetic screening for mutations and polymorphisms, including CF-related mutants and polymorphs, using an array of probe pairs (in one aspect, where one member is complementary to a particular mutant or polymorphic allele and the other member is complementary to a corresponding wild type allele), with probes bound to encoded particles (e.g., beads) wherein the encoding allows identification of the attached probe. The methods relate to avoiding cross-hybridization by selection of probes and amplicons, as well as separation of reactions of certain probes and amplicons where a homology threshold is exceeded. Methods of correcting a fluorescent image using a background map, where the particles also contain an optical encoding system, are also disclosed.
1 - 48 . (canceled)
49 . A method of correcting for false positive signals from mismatched probe-sample binding, or mismatched probe-amplicon binding, based on signals obtained from an oligonucleotide probe array designed to detect genetic mutations or polymorphisms through hybridization of probes to samples, or to amplicons generated from samples, comprising:
forming an array of probes;
contacting said array with said samples, or with said amplicons, under annealing temperature and conditions; heating said contacted array from the annealing temperature through a plurality of set temperature points, each said set temperature point representing the temperature at which a particular mismatched probe-sample hybrid, or a particular mismatched probe-amplicon hybrid, is expected to de-anneal;
monitoring signals from said array during said heating to determine the numbers (or relative numbers) of hybrids at the annealing temperature and at each of said set temperature points; and
interpreting the results from the monitoring step based on the assumption that none of the signals at each respective set temperature point are from mismatched hybrids that were expected to have de-annealed below said respective set temperature point.
50 . The method of claim 49 wherein said signals are from labels attached to said amplicons or samples.
51 . The method of claim 50 wherein the labels can be optically detected.
52 . The method of claim 49 wherein the samples of said mismatched probe-sample hybrids, or the amplicons of said mismatched probe-amplicon hybrids, differ in sequence by one nucleotide from the properly matched samples or amplicons.
53 . The method of claim 49 wherein said set temperature points are within the range of 45° to 60° C.
54 - 61 . (canceled)