(Authors 1, 2): The Cancer Center, University of California, San Francisco
(Author 3): Life Sciences Division, Lawrence Berkeley Laboratory,
(Authors 4, 6): The Cancer Center, University of California, San Francisco, San Francisco, CA.; Life Sciences Division, Lawrence Berkeley Laboratory
(Author 5): Life Sciences Division, Lawrence Berkeley Laboratory
We describe the development of a robotic instrument capable of printing dense arrays of DNA targets for use in comparative genomic hybridization (CGH). P1, BAC or cosmid DNA clones are dissolved in a mixture of water, DMSO and nitrocellulose, sheared, and placed in a 864-microwell plate. The printing head has a square array of 9 spring-loaded fused silica capillaries on 3 mm centers, each connected by flexible tubing to a manifold. The printing tips of the capillaries are ground to sharp points. To effect printing, DNA samples are drawn into the capillaries under a small negative manifold pressure. Next, the pressure is reduced to ambient, the capillaries are removed from the wells, and the manifold is slightly pressurized. The printing head is moved to an accurately indexed position over an xy translation table containing up to 48 aminosilane-derivitized slides. For printing, the print head is lowered repeatedly as the slides are moved under it. At least 200 consecutive spots may be printed from one loading. The size of the target spot depends on the capillary i.d. and tip sharpness. A 365 µm o.d. x 75 µm i.d. capillary sharpened to ~125 µm o.d. produced highly uniform (160±8 µm dia.) circular spots. Similarly prepared 25 µm i.d. capillaries can print spots ~ 50 µm in diameter. This permits printing on ~100 µm centers so that target densities of 10,000/cm2 can be achieved. The reproducibility in target size, position and shape simplifies the data analysis. Use of robotically printed arrays for analysis of copy number changes in breast cancer will be described.