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UWE R. MULLER et al.
- Mix labeled sample and reference DNA with Cot-l DNA to suppress
repeat sequences
- Hybridize to DNA chip and remove unbound probes through washing
- Image chip to determine the fluorescence intensities in each color for
each target spot
- Statistical analysis of color ratios and normalization to determine
relative copy numbers
Procedure
Array fabrication
The arrays employed in this study were fabricated by a novel, highthroughput contact printing technology developed at Corning Incorporated that allows for the simultaneous deposition/printing of )1000 elements within 6 seconds. Briefly, four grids were printed on each GAPS
coated slide, whereby each slide contains 3392 human genes (including
house-keeping genes and positive controls) and several Bacillus subtilus
genes (negative controls).
Genomic DNA labeling
Since DNA extraction is a common practice, it will not be described. Genomic DNA was labeled by primer extension as follows. A 28 III solution
containing 4 Ilg of human genomic DNA and 4 Ilg of random hexamers
was denatured for 5 min at 95°C, briefly chilled on ice, and then combined
with 4111 of 10 x EcoPol buffer, 2 III of 0.1 M DTT, 3 III of a dNTP mixture
(10 mM each dGTP, dATP, dTTP, and 1 mM of dCTP), 2111 ofCy3- or Cy5dCTP (1 mM, PerkinElmer, Boston, MA), and 1 III of Klenow fragment
(New England Biolabs, Inc., Beverly, MA). This resulting 40 III reaction
mixture was incubated at 37°C for two hours. Probes were purified using
a QIAquick PCR purification kit according to the manufacturer's instructions (Qiagen, Inc., Valencia, CA). The DNA concentration and the
amount of Cy3/Cy5 incorporation was measured on the Agilent 8453E
UV-Vis spectrometer. It is important to note that signal intensities will
vary even for target spots that have similar test/reference ratios. This is
most likely due to differences in hybridization kinetics due to variations
UWE R. MULLER et al.
- Mix labeled sample and reference DNA with Cot-l DNA to suppress
repeat sequences
- Hybridize to DNA chip and remove unbound probes through washing
- Image chip to determine the fluorescence intensities in each color for
each target spot
- Statistical analysis of color ratios and normalization to determine
relative copy numbers
Procedure
Array fabrication
The arrays employed in this study were fabricated by a novel, highthroughput contact printing technology developed at Corning Incorporated that allows for the simultaneous deposition/printing of )1000 elements within 6 seconds. Briefly, four grids were printed on each GAPS
coated slide, whereby each slide contains 3392 human genes (including
house-keeping genes and positive controls) and several Bacillus subtilus
genes (negative controls).
Genomic DNA labeling
Since DNA extraction is a common practice, it will not be described. Genomic DNA was labeled by primer extension as follows. A 28 III solution
containing 4 Ilg of human genomic DNA and 4 Ilg of random hexamers
was denatured for 5 min at 95°C, briefly chilled on ice, and then combined
with 4111 of 10 x EcoPol buffer, 2 III of 0.1 M DTT, 3 III of a dNTP mixture
(10 mM each dGTP, dATP, dTTP, and 1 mM of dCTP), 2111 ofCy3- or Cy5dCTP (1 mM, PerkinElmer, Boston, MA), and 1 III of Klenow fragment
(New England Biolabs, Inc., Beverly, MA). This resulting 40 III reaction
mixture was incubated at 37°C for two hours. Probes were purified using
a QIAquick PCR purification kit according to the manufacturer's instructions (Qiagen, Inc., Valencia, CA). The DNA concentration and the
amount of Cy3/Cy5 incorporation was measured on the Agilent 8453E
UV-Vis spectrometer. It is important to note that signal intensities will
vary even for target spots that have similar test/reference ratios. This is
most likely due to differences in hybridization kinetics due to variations