Development of a DNA-labeling system for array-based comparative genomic hybridization.
Chromosomal amplifications and deletions are critical components of tumorigenesis and DNA copy-number variations also correlate with changes in mRNA expression levels. Genome-wide microarray comparative genomic hybridization (CGH) has become an important method for detecting and mapping chromosomal changes in tumors. Thus, the ability to detect twofold differences in fluorescent intensity between samples on microarrays depends on the generation of high-quality labeled probes. To enhance array-based CGH analysis, a random prime genomic DNA labeling method optimized for improved sensitivity, signal-to-noise ratios, and reproducibility has been developed. The labeling system comprises formulated random primers, nucleotide mixtures, and notably a high concentration of the double mutant exo-large fragment of DNA polymerase I (exo-Klenow). Microarray analyses indicate that the genomic DNA-labeled templates yield hybridization signals with higher fluorescent intensities and greater signal-to-noise ratios and detect more positive features than the standard random prime and conventional nick translation methods. Also, templates generated by this system have detected twofold differences in gene copy number between male and female genomic DNA and identified amplification and deletions from the BT474 breast cancer cell line in microarray hybridizations. Moreover, alterations in gene copy number were routinely detected with 0.5 microg of genomic DNA starting sample. The method is flexible and performs efficiently with different fluorescently labeled nucleotides. Application of the optimized CGH labeling system may enhance the resolution and sensitivity of array-based CGH analysis in cancer and medical genetic studies.
Lieu
Pauline T
PT
Invitrogen Life Technologies, 1610 Faraday Ave, Carlsbad, CA 92008, USA. Pauline.lieu@Invitrogen.com
Jozsi
Peter
P
Gilles
Patrick
P
Peterson
Todd
T
D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000235:genetics / Q000378:metabolism*; D005260:Female; D023281:Genomics; D006801:Humans; D008297:Male; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D013194:Staining and Labeling
D004273:DNA, Neoplasm