Optimization of DOP‐PCR amplification of DNA for high‐resolution comparative genomic hybridization analysis

• Published in: Cytometry (New York, N.Y.) Vol. 44; no. 4; pp. 317 - 325
• Main Authors: Larsen, Jacob, Ottesen, Anne Marie, Lundsteen, Claes, Leffers, Henrik, Larsen, Jørgen K.
• Format: Journal Article
• Language: English
• Published: New York John Wiley & Sons, Inc 01.08.2001
• Subjects: Breast Neoplasms - genetics; comparative genomic hybridization; DNA Primers - genetics; DNA, Neoplasm - analysis; DOP‐PCR; Humans; Leukemia, Myeloid - genetics; Nucleic Acid Amplification Techniques - methods; Nucleic Acid Amplification Techniques - standards; Nucleic Acid Hybridization - genetics; Nucleic Acid Hybridization - methods; PCR; Polymerase Chain Reaction - methods; Tumor Cells, Cultured
• ISSN: 0196-4763; 1097-0320
• DOI: 10.1002/1097-0320(20010801)44:4<317::AID-CYTO1123>3.0.CO;2-5

Background Whole‐genome amplification of minute samples of DNA for the use in comparative genomic hybridization (CGH) analysis has found widespread use, but the method has not been well validated. Methods Four protocols for degenerate oligonucleotide primed polymerase chain reaction (DOP‐PCR) and fluorescence labeling were applied to test DNA from normal and K‐562 cells. The DNA products were used for CGH analysis. Results The DOP‐PCR–amplified DNA from each protocol produced hybridizations with different qualities. These could be seen primarily as differences in background staining and signal‐to‐noise ratios, but also as characteristic deviations of normal/normal hybridizations. One DOP‐PCR‐protocol was further investigated. We observed concordance between CGH results using unamplified and DOP‐PCR–amplified DNA. An example of an analysis of an invasive carcinoma of the breast supports the practical value of this approach. Conclusions DOP‐PCR–amplified DNA is applicable for high‐ resolution CGH, the results being similar to those of CGH using unamplified DNA. Cytometry 44:317–325, 2001. © 2001 Wiley‐Liss, Inc.