Methods for detecting aneuploidy using microparticle multiplex detection

Abstract

The present invention provides a method for the detection and sorting of microparticles in a mixture of microparticles. The method of the present invention allows for the detection and sorting of many distinct microparticle classes. Detection and sorting is on the basis of microparticle size, the fluorescence spectrum of any attached reporter molecule, the fluorescence intensity of the reporter molecule, and the number of particles in each classification bin. These microparticle classes have particular applications in many genetic or biochemical multiplexing studies and especially as binding agents for the detection of aneuploidy in an organism or embryo of the organism. In humans, the detection and sorting of at least 24 classes of microparticles would be sufficient for a single tube method for the simultaneous detection of aneuploidy in all chromosomes, wherein each distinct microparticle class comprises a polynucleotide sequence complementary to, and specific for, a polynucleotide sequence that is unique to a particular human chromosome. Furthermore, using currently available technology, the present method has application for the simultaneous detection of aneuploidy in all chromosomes for an organism that has 216 or fewer pairs of chromosomes. Kits for the simultaneous detection of aneuploidy in one or more human chromosomes are also provided.

1 . A method for detecting aneuploidy in one or more chromosomes of a subject simultaneously, said method comprising:
 (i) producing fluorescently-labelled polynucleotide samples that are representative of the abundance of each chromosome in said subject;   (ii) further producing equivalent, non-aneuploid polynucleotide standards for each chromosome, labelled with a different fluorescent marker to the sample;   (iii) mixing said sample and standard with a limiting amount of binding agents for each chromosome, wherein said binding agents comprise a polynucleotide that is complementary to the sample and standard for each chromosome immobilized onto a microparticle, and the microparticles for each chromosome are distinct on the basis of size and/or fluorescent label and/or fluorescent label intensity;   
       wherein the fluorescent label on the microparticle, if present, has a distinct emission spectrum from both the label of the sample and standard; and wherein aneuploidy is detected as non-equal binding of said sample and said standard to said binding agent. 
     
     
         2 . The method according to  claim 1 , wherein the patient is a diploid organism. 
     
     
         3 . The method of  claim 2 , wherein the subject is a mammal. 
     
     
         4 . The method according to any one of  claims 1  to  3  wherein said mammal is a human. 
     
     
         5 . The method according to any one of  claims 1  to  3  wherein the animal is a livestock animal. 
     
     
         6 . The method according to  claim 5 , wherein the livestock animal is selected from cattle, sheep and horses. 
     
     
         7 . The method according to any one of  claims 1  to  6 , wherein the subject is an embryo. 
     
     
         8 . The method of  claim 7  wherein said embryo is generated using in-vitro fertilization. 
     
     
         9 . The method according to  claim 7  or  8 , wherein said method is suitable for the detection of aneuploidy in said embryo prior to implantation. 
     
     
         10 . The method according to  claim 9 , wherein the DNA sample is isolated, generated or amplified from a blastomere. 
     
     
         11 . The method according to any one of  claims 1  to  6  wherein the nucleic acid sample and standard are produced from genomic DNA from a somatic cell. 
     
     
         12 . The method according to any one of  claims 1  to  6  wherein the nucleic acid sample and/or standard are produced from genomic DNA from a reproductive cell or gamete. 
     
     
         13 . The method of any one of  claims 1  to  12  wherein said binding agent comprises a nucleic acid, with binding specificity for the sample and standard, immobilized on a microparticle. 
     
     
         14 . The method according to  claim 13  wherein the microparticle is a silica microparticle. 
     
     
         15 . The method according to  claim 14 , wherein the silica microparticle is silanized. 
     
     
         16 . The method of any one of  claims 1  to  15  wherein the labelled sample and/or standard, and/or relative amounts of labelled sample to standard, are determined using a flow cytometer. 
     
     
         17 . A kit for the simultaneous diagnosis of aneuploidy in one or more chromosomes in an organism, embryo or cell, comprising:
 (i) fluorescently labelled oligonucleotide primer sets suitable for the amplification of chromosome specific polynucleotide sequences;   (ii) duplicate sets of oligonucleotide primers with identical sequence to the first sets, but comprising a different fluorescent marker with a distinct emission spectrum to the first marker;   (iii) a number of binding agents, distinct on the basis of microparticle size, reporter molecule, or reporter molecule intensity, equal to the number of chromosomes in the subject, wherein each binding agent comprises a polynucleotide sequence complementary to the predicted amplicon of the oligonucleotide primers which is immobilized to a labelled or unlabelled microparticle;   (iv) instructions for the use of said reagents;   
       wherein the label of the microparticle, if present has a distinct emission spectrum to the label of the both labelled oligonucleotide primers.