SUBSTRACTIVE SINGLE LABEL COMPARATIVE HYBRIDIZATION
First Claim
1. A method of determining genomic abnormalities in a test sample of genomic nucleic acid, comprising:
- amplifying genomic sequence from a test sample and amplifying genomic sequence from a reference sample nucleic acid, wherein one of the amplification reactions is conducted using dUTP and not dTTP and the other is conducted using dTTP and not dUTP;
hybridizing to a genomic nucleic acid array a solution comprising the amplified test sample and amplified reference sample; and
determining the relative amount of hybridized test and reference nucleic acids bound to the array, wherein a difference in the relative amount of hybridized test and reference nucleic acids bound to the array identifies the presence of a genomic abnormality.
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Abstract
Provided are methods of determining differences between nucleic acids in a test sample and a reference sample. In certain embodiments the methods are used for 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, provided are 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.
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Citations
22 Claims
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1. A method of determining genomic abnormalities in a test sample of genomic nucleic acid, comprising:
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amplifying genomic sequence from a test sample and amplifying genomic sequence from a reference sample nucleic acid, wherein one of the amplification reactions is conducted using dUTP and not dTTP and the other is conducted using dTTP and not dUTP; hybridizing to a genomic nucleic acid array a solution comprising the amplified test sample and amplified reference sample; and determining the relative amount of hybridized test and reference nucleic acids bound to the array, wherein a difference in the relative amount of hybridized test and reference nucleic acids bound to the array identifies the presence of a genomic abnormality. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method of determining differences between nucleic acid in a test sample and a reference sample is provided said method comprising:
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(a) contacting under hybridization conditions a test sample containing nucleic acids and a reference sample containing nucleic acids to a surface containing a plurality of nucleic acid segments each immobilized at discrete locations on the surface, wherein the test sample and the reference sample are labeled before or after hybridization with the same detectable label; (b) determining the location and amount of the detectable label linked to nucleic acids hybridized to the surface; (c) selectively removing either the hybridized test sample genomic nucleic acids or the hybridized reference sample genomic nucleic acids; (d) determining the location and amount of the detectable label linked to nucleic acids hybridized to the surface following step (c); and (e) comparing the results of step (b) to the results of step (d) to detect differences in the nucleic acids of the test sample and reference sample. - View Dependent Claims (17, 18, 19, 20, 21)
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22. A method of comparing the expression of genes in a test sample versus that of a reference sample said method comprising
amplifying nucleic acid sequence from cDNA prepared from RNA of the test sample and amplifying nucleic acid sequence from cDNA prepared from RNA of the reference sample, wherein one of the amplification reactions is conducted using dUTP and not dTTP and the other is conducted using dTTP and not dUTP; -
hybridizing to the nucleic acid array a solution comprising the amplified test sample and amplified reference sample; performing a first scan to determine a signal for the detectable label hybridized to the array representing the total of hybridized test and reference nucleic acid; treating the hybridized nucleic acids with an enzyme that selectively degrades DNA having uracil residues; performing a second scan to determine a signal for the detectable label hybridized to the array following said treating the hybridized nucleic acids with an enzyme that selectively degrades DNA having uracil residues; and comparing the signal from said first scan with the signal from said second scan to determine differences between said test sample and said reference sample in the expression of at least one gene.
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Specification