Single label comparative hybridization

Abstract

The present invention provides methods of detecting and mapping chromosomal or genetic abnormalities associated with various diseases or with predisposition to various diseases, or to detecting the phenomena of large scale copy number variants. In particular, the present invention provides advanced methods of performing array-based comparative hybridization that allow reproducibility between samples and enhanced sensitivity by using the same detectable label for both test sample and reference sample nucleic acids. Invention methods are useful for the detection or diagnosis of particular disease conditions such as cancer, and detecting predisposition to cancer based on detection of chromosomal or genetic abnormalities and gene expression level. Invention methods are also useful for the detection or diagnosis of hereditary genetic disorders or predisposition thereto, especially in prenatal samples. Moreover, invention methods are also useful for the detection or diagnosis of de novo genetic aberrations associated with post-natal developmental abnormalities.

1 - 69 . (canceled) 
     
     
         70 . A method of detecting a chromosomal abnormality in a test sample of genomic nucleic acid, comprising:
 (i) simultaneously hybridizing a test genomic nucleic acid sample and a reference genomic nucleic acid sample to a nucleic acid array wherein the array comprises a plurality of individual spots, wherein each spot comprises a plurality of substantially identical nucleic acid probes;   (ii) after step (i), comparing the relative amount of test genomic nucleic acid and reference genomic nucleic acid at a plurality of individual spots as an indication of the comparative hybridization, wherein the test and reference genomic nucleic acid are labeled with an indistinguishable detectable label and a signal from the indistinguishable detectable label is used to determine the amount of hybridization of the test genomic nucleic acid and reference genomic nucleic acid to an individual spot; and   (iii) determining the presence of a chromosomal abnormality based on the difference in the amount of the test genomic nucleic acid and the reference genomic nucleic acid hybridized to the nucleic acid array.   
     
     
         71 . The method of  claim 70 , wherein the chromosomal abnormality is a chromosomal translocation, a change in chromosomal copy number, a chromosomal deletion, a chromosomal amplification, or a chromosomal mutation. 
     
     
         72 . The method of  claim 70 , wherein the indistinguishable detectable label is attached to the test and reference genomic nucleic acids before hybridization. 
     
     
         73 . The method of  claim 70 , wherein the indistinguishable detectable label is attached to the test and reference genomic nucleic acids after hybridization. 
     
     
         74 . The method of  claim 70 , wherein the indistinguishable detectable label is attached to the test and reference genomic nucleic acids via a ligand-receptor interaction or by hybridization between complementary nucleotide sequences. 
     
     
         75 . The method of  claim 70 , wherein one of the test or reference genomic nucleic acids is associated with the indistinguishable detectable label during hybridization and the detectable label is attached to the other of the test or reference genomic nucleic acids after hybridization. 
     
     
         76 . The method of  claim 70  wherein the amount of hybridized test and reference genomic nucleic acids are determined by:
 a) determining a signal representing one of the test or reference genomic nucleic acid hybridized to the plurality of individual spots by detecting a first complex comprising a label and a first entity, wherein the first complex selectively binds to one of the test or reference genomic nucleic acid via a first tag attached to the test or reference genomic nucleic acid, the first entity and tag being members of a specific binding pair; 
 b) determining a signal representing the total of the hybridized test and reference genomic nucleic acid to each individual spot by contacting the hybridized array of step a) with a second complex comprising the label of step a) and a second entity, wherein the second complex selectively reacts with the other of the test or reference genomic nucleic acid via a second tag attached to the other of the test or reference genomic nucleic acid detected in step b), the second entity and second tag being members of a specific binding pair; 
 c) using the signal from a) and b) to obtain the signal for the other of the hybridized test or reference genomic nucleic acid not detected in step a); and 
 d) comparing the amount of hybridization of the test and reference genomic nucleic acids to the plurality of individual spots. 
 
     
     
         77 . The method of  claim 70  wherein the amount of hybridized test and reference nucleic acids are determined by:
 a) determining a signal representing one of the test or reference genomic nucleic acid hybridized to the plurality of individual spots by detecting a first complex comprising a label and a first entity, wherein the first complex selectively binds to one of the test or reference genomic nucleic acid via a first tag attached to the test or reference genomic nucleic acid, the first entity and tag being members of a specific binding pair; 
 b) determining a signal representing the total of the hybridized test and reference genomic nucleic acid to each individual spot by contacting the hybridized array of step a) with a second complex comprising the label of step a) and a second entity, wherein the second complex selectively reacts with the other of the test or reference genomic nucleic acid via a second tag attached to the other of the test or reference genomic nucleic acid detected in step b), the second entity and second tag being members of a specific binding pair; 
 c) using the signal from a) and b) to obtain the signal for the other of the hybridized test or reference genomic nucleic acid not detected in step a); and 
 d) comparing the amount of hybridization of the test and reference nucleic acids to the plurality of individual spots. 
 
     
     
         78 . The method of  claim 77 , wherein the first complex is bound to the test or reference genomic nucleic acid detected in a) after hybridization to the array. 
     
     
         79 . The method of  claim 77 , wherein the first complex is bound to the test or reference genomic nucleic acid detected in a) before hybridization to the array. 
     
     
         80 . The method of  claim 77 , wherein the first tag and the second tag each comprises a unique oligonucleotide capture sequence. 
     
     
         81 . The method of  claim 80 , wherein the first complex and the second complex each comprises an oligonucleotide complementary to one of the unique oligonucleotide capture sequences. 
     
     
         82 . The method of  claim 70 , wherein the nucleic acid array comprises a plurality of nucleic acid segments each contained within an artificial chromosome. 
     
     
         83 . The method of  claim 70 , wherein the nucleic acid array comprises a plurality of nucleic acid segments, each nucleic acid segment of the plurality being between about 1,000 and about 1,000,000 nucleotides in length. 
     
     
         84 . The method of  claim 83 , wherein the plurality of nucleic acid segments comprise segments of chromosomal sequence of about 3-4 megabases in length, about 2-3 megabases in length, or about 1-2 megabases in length. 
     
     
         85 . The method of  claim 70 , wherein the detectable label is a fluorophore. 
     
     
         86 . The method of  claim 75 , wherein the first linkage and the second linkage differ in susceptibility to temperature, pH, hydrolysis, radiation, change in oxidative conditions, change in atmospheric conditions, change in buffer conditions, or enzymatic cleavage. 
     
     
         87 . The method of  claim 86 , wherein the first linkage or the second linkage comprises an oligonucleotide containing a recognition site for a restriction endonuclease or a homing endonuclease. 
     
     
         88 . The method of  claim 70 , wherein the test sample from a patient is suspected of having cancer. 
     
     
         89 . The method of  claim 70 , wherein the test sample is a prenatal specimen.