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  • Poster Presentation
  • Open Access

Comparative genomic hybridization using oligonucleotide arrays and total genomic DNA

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Breast Cancer Research20057 (Suppl 2) :P4.40

https://doi.org/10.1186/bcr1170

  • Published:

Keywords

  • National Human Genome Research Institute
  • Copy Number Polymorphism
  • Adenocarcinoma Cell Line HT29
  • Colon Adenocarcinoma Cell Line HT29
  • Polymerase Phi29

Array-based comparative genomic hybridization (aCGH) measures copy number variations at multiple loci simultaneously, providing an important tool for studying genomic alterations associated with cancer, developmental disorders, and germline copy number polymorphisms. The broadest utility of aCGH is obtained by enabling flexible and high-resolution probing of regions of interest while preserving the greatest possible complexity of targets derived from whole genome samples. We therefore developed probe design criteria, assay conditions, and analysis methods that enable 60-mer oligonucleotide arrays to be used for CGH measurements using total genomic DNA [1]. We designed a 60-mer oligonucleotide array with 40K probes specifically designed for CGH representing sequences throughout the human genome with a bias for known and predicted gene loci. We tested the performance of this array for reproducibly measuring and mapping losses, and amplification events of varying levels and sizes using both unamplified and phi29 (Qiagen, Valenica, CA, USA) amplified total genomic DNA from a series of model systems. The mean slope of experimental versus theoretical log-ratios for chromosome X probes on this genome-wide human CGH array in XY versus XX hybridizations typically exceeds 0.9, with probe by probe error rates of less than 10% in the separation of their log-ratio distributions. Additionally, we used this platform to examine well-characterized cell lines, including diploid cells with partial deletions in chromosome 18q, and diploid and aneuploid tumor cell lines with known amplification and deletion events. We show that the highly processive DNA polymerase phi29 can be used to prepare aCGH templates from as little as 10 ng starting material that yield high-quality aCGH measurements throughout the genome. While phi29 provides a simplified isothermal method for amplifying limiting material, non-specific DNA fragments of high MW are generated in the absence of sufficient input template. Although these products do not hybridize to the array, the presence of these amplification products obscures the accurate quantification of DNA template specific to the input genomic DNA prior to the labeling reaction. To ensure reproducible and robust aCGH assay quality, we developed methods and protocols using the Agilent BioAnalyzer (Agilent Technologies, Palo Alto, CA, USA) to enable accurate quality control for key prehybridization steps, including: phi29 amplification of genomic samples, restriction digestion of templates and target labeling. We have also developed visualization tools and statistically robust computational tools that take into account the estimated errors on the measured log ratios in mapping aberration boundaries, and for identifying common aberrations across multiple samples. We tested the reproducibility of our platform using tumor cell line samples including the colon adenocarcinoma cell line HT29 in hybridizations performed in different laboratories (Agilent Labs, National Human Genome Research Institute, Translational Genomics Institute). We present results, using these methods, demonstrating that in situ synthesized 60-mer oligonucleotide arrays can reproducibly detect genomic lesions including single copy and homozygous deletions, and variable amplicons throughout the genome using full complexity genomic DNA samples.

Authors’ Affiliations

(1)
Agilent Technologies, Palo Alto, California, USA
(2)
Translational Genomics Institute, Phoenix, Arizona, USA
(3)
National Human Genome Research Institute, Bethesda, Maryland, USA

References

  1. Barrett MT, Scheffer A, Ben-Dor A, Sampas N, Lipson D, Kincaid R, Tsang P, Curry B, Baird K, Meltzer PS, et al: Comparative genomic hybridization using oligonucleotide microarrays and total genomic DNA. Proc Natl Acad Sci USA. 2004, 101: 17765-17770. 10.1073/pnas.0407979101.View ArticlePubMedPubMed CentralGoogle Scholar

Copyright

© BioMed Central 2005

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