Hybridization-Mediated Analysis Of Polymorphisms

Abstract

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 .- 61 . (canceled) 
     
     
         62 . An encoded particle composition comprising
 a polystyrene bead with a diameter of between 0.2 and 200 microns
 wherein the bead is magnetic 
 and is stained with two or more fluorophores,
 wherein the fluorophores are distinguishable by emission wavelength and generate an optical signature; and 
 
   an oligonucleotide probe with a length of 15-25 bases
 wherein the sequence of the probe is selected to bind to a labeled amplicon generated from a genomic sample, and 
 wherein the probe is attached to the bead via its 5′ terminus; and 
   
       wherein the optical signature uniquely corresponds to the probe. 
     
     
         63 . The composition of  claim 62 , wherein the bead is labeled with two fluorophores. 
     
     
         64 . The composition of  claim 62 , wherein the sequence of the probe is complementary to a mutant allele, a polymorphic allele, or a wild-type allele. 
     
     
         65 . A method of forming a planar array of beads, comprising
 subjecting particles according to  claim 62  to an electromagnetic field.