SUBTRACTIVE SINGLE LABEL COMPARATIVE HYBRIDIZATION
First Claim
1. A method of determining a nucleotide sequence difference between nucleic acid in a test sample and nucleic acid in a reference sample, comprising:
- amplifying nucleic acid from a test sample and amplifying nucleic acid from a 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 a 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 nucleotide sequence difference.
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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
19 Claims
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1. A method of determining a nucleotide sequence difference between nucleic acid in a test sample and nucleic acid in a reference sample, comprising:
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amplifying nucleic acid from a test sample and amplifying nucleic acid from a 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 a 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 nucleotide sequence difference. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification