Technical Approaches for Efficient, High Precision Nucleic Acid Analysis Using DNA Microarrays.

摘要

Microarray measurements offer the potential to compare the abundances of numerous nucleic acid sequences in parallel. Using linker-adapter PCR products from mapped BAC clones we have made arrays that permit scanning the human genome for single copy gains and losses of DNA sequence, which requires reliable detection of 50% changes. The DNA is printed at high concentration on amino-silane or chromium coated surfaces using a custom-built capillary pin printing system. Spots are printed on 130 μm centers or closer to minimize the size of the arrays. Hybridization occurs in a dextran sulfate/formamide buffer at 37 °C, using slow rocking to mix the reaction. The entire array is imaged in a single CCD frame using a custom built system that employs mercury arc illumination. Up to four fluorochromes can be imaged from a single array with adequate spectral separation. Typically we used DAPI to stain the DNA in the array spots to facilitate automatic image segmentation during analysis, and fluorescein, Cy3, and Cy5 or their spectral equivalents, for labeling specimen nucleic acids. Array spots are segmented and quantitative fluorescence intensities and intensity ratios are automatically calculated in <1 minute per ～8000 element array using the custom software UCSF SPOT.