Hybridization-mediated analysis of polymorphisms
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 - 15 . (canceled)
16 . A method of optimizing a hybridization assay for detecting known genetic mutations and polymorphisms in genomic targets, comprising the following:
a) providing a set of oligonucleotide primer pairs capable of amplifying a region of said genomic targets that includes at least one designated mutation or polymorphic site
to thereby generate a set of amplicon pairs, each amplicon pair comprising a first member having a region complementary to a subsequence of the genomic target coding strand and a second member having a region complementary to a subsequence of the genomic target antisense strand;
b) selecting two groups of encoded probes wherein probes that are encoded differently have different nucleotide sequences, sense probes selected such that each sense probe is complementary, in whole or in substantial part, to a region of a designated first member, and antisense probes selected such that each antisense probe is complementary, in whole or in substantial part, to a region of a designated second member; and
c) reducing hybridization between particular amplicons with more than one probe by selecting primer pairs to reduce the number of said regions is generated on a particular amplicon.
17 . The method of claim 16 further including determining the number of said regions in an amplicon member designated for hybridization with a particular probe, removing amplicon members from the set that include more than one region, and replacing said removed amplicon members with other amplicon members having fewer said regions.
18 - 61 . (canceled)
62 . The method of claim 16 wherein the genomic target is an mRNA, a cDNA or a double-stranded DNA.
63 . The method of claim 16 wherein probes having different sequences are encoded by linking probes to microparticle carriers, including beads, said carriers having different optical signatures.
64 . The method of claim 16 wherein the encoding is with color.
65 . The method of claim 16 wherein one primer in a primer pair is modified at the 5′ end with a label and the other primer in the primer pair has a phosphate modification at the 5′ end.
66 . The method of claim 65 wherein the amplicon incorporating said phosphate-modified primer is digested.
67 . The method of claim 16 wherein the hybridization of members and probes is determined by detecting signals from the labels associated with said members.