Multiple reporter read-out for bioassays

A method for determining allele zygosity of nucleic acid molecules of a genetic locus having two alleles, the method comprising:

• (1) providing a population of microspheres, wherein each of the microspheres carries two different nucleic acid probes respectively specific to each of the two alleles;
• (2) allowing the probes to hybridize to the two alleles, thereby forming allele-probe pairs on the microspheres, wherein different allele-probe pairs are detectably distinguishable by their fluorescence intensity, and wherein the different allele-probe pairs are not detectably distinguishable by their fluorescence wavelength;
• and
• (3) detecting the fluorescence intensity of the allele-probe pairs formed on one or the microspheres simultaneously, whereby the allele zygosity of the genetic locus is determined based on the fluorescence intensity of the allele-probe pairs.

A method for determining polymorphism of nucleic acid molecules of a genetic locus having multiple alleles, the method comprising:

• (1) providing a population of microspheres, wherein each of the microspheres carries multiple different nucleic acid probes respectively specific to each of the multiple alleles;
• (2) allowing the probes to hybridize to the multiple alleles, thereby forming allele-probe pairs on microspheres, wherein different allele-probe pairs are distinguishable by fluorescence intensity, and wherein the different allele-probe pairs are not distinguishable by fluorescence wavelength;
• and
• (3) detecting the fluorescence intensity of the allele-probe pairs formed on one of the microspheres simultaneously, whereby the allele polymorphism of the genetic locus is determined based on the fluorescence intensity of the allele-probe pairs.

A method for detecting different analytes in a sample, the method comprising:

• (1) providing a population of microspheres, wherein each of the microspheres carries different reactants capable of reacting respectively to each of the different analytes;
• (2) allowing the reactant and the analytes to react, thereby forming reactant-analyte pairs;
• (3) providing a mixture of reporter reagents capable of reacting with the reactant-analyte pairs;
• (4) allowing the reporter reagents to react with the reactant-analyte pairs to form reactant-analyte-reporter reagent complexes, wherein different reactant-analyte-reporter reagent complexes are distinguishable by fluorescence intensity, but not by fluorescence wavelength, wherein the analytes are alleles of target nucleic acid molecules of a genetic locus, wherein the reactants are capture probes complementary to a common sequence within the genetic locus, and wherein the reporter reagents are reporter probes specific to the individual alleles;
• and
• (5) detecting the fluorescence intensity of the reactant-analyte-reporter reagent complexes formed on one of the microspheres simultaneously, whereby the presence or absence of each of the different analytes is determined based on the fluorescence intensity.

A method for detecting SNPs in target nucleic acid molecules, wherein each of the SNPs has two or more polymorphisms, the method comprising:

• (1) providing a plurality of populations of microspheres, wherein each population corresponds to one of the SNPs and has an addressable signature, and wherein each of the microspheres in a population carries two or more nucleic acid probes specific respectively to each of the polymorphisms for the SNP;
• (2) allowing the probes to hybridize to the SNPs, thereby forming SNP-probe pairs, wherein different SNP-probe pairs are distinguishable by fluorescence intensity but not by fluorescence wavelength;
• and
• (3) determining the type of SNPs via detecting the fluorescence intensity of the SNP-probe pairs formed on one of the microspheres simultaneously, and detecting the addressable signature.

A method for detecting different reactive sites on an analyte, comprising:

• allowing different reactants to react with the different reactive sites on the analyte to form one or more reactant-reactive site pairs, wherein the different reactants are carried on a microsphere, and wherein each of the different reactants is capable of reacting specifically with one of the different reactive sites;
• analyzing the microsphere to detect output signal responsive to each of the reactant-reactive site pairs, formed on the microsphere, wherein different reactant-reactive site pairs are distinguishable by their fluorescence intensity but not by their fluorescence wavelength;
• determining which of the reactant-reactive site pairs were formed using the output signal;
• and
• determining which of the different reactive sites are present on the analyte using results of said determining, wherein a determination that a reactant-reactive site pair was formed indicates a presence of the reactive site, corresponding to the formed reactant-reactive site pair, on the analyte.

A method for detecting different analytes in a sample, comprising:

• allowing different reactants to react with the different analytes to form one or more reactant-analyte pairs, wherein the different reactants are carried on a microsphere, and wherein each of the different reactants are capable of reacting specifically with one of the different analytes;
• allowing reporter reagents to react with the one or more reactant-analyte pairs to form reactant-analyte-reporter reagent complexes;
• analyzing the microsphere to detect an output signal responsive to each of the complexes formed on the microsphere, wherein different complexes are distinguishable by their fluorescence intensity but not by their fluorescence wavelength;
• determining which of the complexes were formed using the output signal;
• and
• determining which of the different analytes are present in the sample using results of said determining, wherein a determination that a complex was formed indicates a presence of the analyte, corresponding to the formed complex, in the sample.