Differentiating homozygous, heterozygous and wild-type alleles using a multiplexed hybridization-mediated assay
View Patent ↗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. A method of differentiating homozygous, heterozygous and wild-type alleles in a target sample comprising mutant or polymorphic alleles or wild-type alleles using results obtained from a probe array, wherein individual probes in the array are designed to detect designated mutant or polymorphic alleles or wild-type alleles through hybridization of said probes and targets, where the results compensate for hybridization between probes and targets other than their designated targets, comprising:
amplifying the genomic regions in the target sample that are predicted to include said mutant or polymorphic alleles or said wild-type alleles, thereby producing a first set of labeled amplicons having a region complementary to a subsequence of said mutant or polymorphic alleles (if said mutant or polymorphic alleles are present in said target sample) and a second set of labeled amplicons having a region complementary to a subsequence of said wild-type alleles (if said wild-type alleles are present in said target sample);
providing an array of probe pairs, the first member of each pair being complementary, in whole or in substantial part, to a subsequence of a first-set amplicon and the second member of each pair being complementary, in whole or in substantial part, to a subsequence of a second-set amplicon;
contacting under hybridizing conditions said array of probe pairs with said amplicons;
detecting hybridization between probes and said amplicon sets based on the presence of signals from the labeled, hybridized amplicons, said signals being corrected to adjust for hybridization between probes and amplicons other than their designated amplicons as follows:
(i) determining the intensity of signals from hybridization of first-set amplicons and from hybridization of second-set amplicons, as corrected for background signals, and
(ii) determining the ratio of said signals, wherein the ratio of the intensity of first-set amplicon signals to the intensity of second-set amplicon signals is designated as a first ratio (a), and wherein the ratio of the intensity of second-set amplicon signals to the intensity of first-set amplicon signals is designated as second ratio (b); and
defining three relative ranges of values for said ratios such that: (i) the lowest range of ratio (a) indicates that said target sample is homozygous for wild-type alleles and the lowest range of ratio (b) indicates that said target sample is homozygous for mutant or polymorphic alleles, (ii) a middle range of either ratio (a) or (b) indicates that said target sample is heterozygous, and (iii) the highest range of ratio (a) indicates that the said target sample is homozygous for mutant or polymorphic alleles and the highest range of ratio (b) indicates that said target sample is homozygous for wild-type alleles.
2. The method of claim 1 further including the step of generating single-stranded DNA from said amplicons.
3. The method of claim 2 further including the step of labeling one of the strands of either of said amplicon sets.
4. The method of claim 1 wherein said relative value ranges are defined such that: >2 indicates that said target sample is homozygous for mutant or polymorphic alleles, <0.5 indicates that said target sample is homozygous for wild-type alleles and 0.8 to 1.2 indicates that said target sample is heterozygous.