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US 12,442,031 B1Granted
DNA labeling compositions and methods for comparative genomic hybridization
AssigneeEnzo Biochem, Inc.
InventorsPraveen Pande, Jack Coleman, Christian Merz
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Abstract
The invention provides compositions and methods for preparing high-quality labeled DNA for use in array-based comparative genomic hybridization in amounts sufficient therefor starting with a low amount of input genomic DNA, such as 50-250 nanograms input genomic DNA.
Claims12
A method for preparing fluorescently labeled output DNA suitable for array-based comparative genomic hybridization from input genomic DNA, comprising the steps of:
- providing a sample of genomic DNA in an amount of 50+/−25% nanograms;
- admixing the sample of genomic DNA with random DNA primers in a reaction buffer comprising magnesium,
- wherein the admixture does not contain either of ethylenediaminetetraacetic acid (EDTA) and egtazic acid (EGTA);
- heating the admixture to denature the genomic DNA and then cooling the admixture to permit the random DNA primers to anneal to the genomic DNA in the admixture;
- further admixing to form a primer extension composition comprising
- a Klenow fragment DNA-dependent DNA polymerase,
- 0.4 mM+20% dATP,
- 0.4 mM+20% dCTP,
- 0.4 mM+20% dGTP,
- 0.4 mM+20% dTTP,
- 0.08 mM+20% to 0.16 mM+20% fluorescently labeled dUTP,
- wherein the primer extension composition formed does not contain either of ethylenediaminetetraacetic acid (EDTA) and egtazic acid (EGTA);
- and
- incubating the primer extension composition at a temperature permissive for primer extension by the polymerase to extend the random DNA primers on the genomic DNA, thereby forming fluorescently labeled DNA extension products.
11 more elements
A method for preparing fluorescently labeled output DNA suitable for array-based comparative genomic hybridization from input genomic DNA, comprising the steps of:
- providing a sample of genomic DNA in an amount of 50+/−25% nanograms;
- admixing the sample of genomic DNA with random DNA primers in a reaction buffer comprising magnesium,
- wherein the admixture does not contain either of ethylenediaminetetraacetic acid (EDTA) and egtazic acid EGTA;
- heating the admixture to denature the genomic DNA and then cooling the admixture to permit the random DNA primers to anneal to the genomic DNA in the admixture;
- further admixing to form a primer extension composition comprising
- a Klenow fragment DNA-dependent DNA polymerase,
- 0.4 mM±20% dATP,
- 0.4 mM±20% dCTP,
- 0.4 mM±20% dGTP,
- 0.2 mM±20% to 0.3 mM±20% dTTP, and
- 0.1 mM±20% to 0.2 mM±20% fluorescently labeled dUTP,
- wherein the primer extension composition formed does not comprise a metal chelating agent;
- and
- incubating the primer extension composition at a temperature permissive for primer extension by the polymerase to extend the random DNA primers on the genomic DNA, thereby forming fluorescently labeled DNA extension products.
11 more elements
Dependencies are inferred from the claim text.