DNA labeling compositions and methods for comparative genomic hybridization
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.
1. 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.
2. The method of claim 1 , wherein the fluorescently labeled dUTP is Cyanine-3 dUTP and its concentration in the primer extension composition is 0.16 mM+20%.
3. The method of claim 1 , wherein the fluorescently labeled dUTP is Cyanine-5 dUTP and its concentration in the primer extension composition is 0.08 mM+20%.
4. The method of claim 1 , further comprising the steps of stopping the primer extension; and
purifying the DNA present to obtain purified DNA comprising the fluorescently labeled DNA extension products.
5. The method of claim 4 , further comprising the step of:
hybridizing the purified DNA comprising the fluorescently labeled DNA extension products to a comparative genomic hybridization (CGH) array.
6. 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.
7. The method of claim 6 , wherein the concentrations of labeled and unlabeled dNTPs in the primer extension composition are:
0.4 mM±20% dATP;
0.4 mM±20% dCTP;
0.4 mM±20% dGTP;
0.25 mM±20% dTTP; and
0.2 mM±20% Cyanine-3 dUTP.
8. The method of claim 6 , wherein the concentrations of labeled and unlabeled dNTPs in the primer extension composition are:
0.4 mM±20% dATP;
0.4 mM±20% dCTP;
0.4 mM±20% dGTP;
0.25 mM±20% dTTP; and
0.2 mM±20% Cyanine-5 dUTP.
9. The method of claim 1 , wherein the random DNA primers comprise random DNA octomers.
10. The method of claim 6 , wherein the random DNA primers consist essentially of random DNA octomers.
11. The method of claim 6 , further comprising the steps of:
stopping the primer extension; and
purifying the DNA present to obtain purified DNA comprising the fluorescently labeled DNA extension products.
12. The method of claim 11 , further comprising the step of:
hybridizing the purified DNA comprising the fluorescently labeled DNA extension products to a comparative genomic hybridization (CGH) array.