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Nucleic Acids Research Advance Access published online on October 1, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp761
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© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Genome Integrity, Repair and Replication

PCR-free method detects high frequency of genomic instability in prostate cancer

Nick M. Makridakis1,*, Troy Phipps2, Sudesh Srivastav3 and Juergen K. V. Reichardt4

1Department of Epidemiology, Tulane University, New Orleans, LA 70112, 2Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, CA 90033, 3Department of Biostatistics, Tulane University, New Orleans, LA 70112, USA and 4Plunkett Chair of Molecular Biology (Medicine), University of Sydney, Camperdown, NSW 2006, Australia

*To whom correspondence should be addressed. Tel: 504-9885418; Fax: 504-9885516; Email: nmakrida{at}tulane.edu

Received May 5, 2009. Revised August 28, 2009. Accepted August 31, 2009.

Most studies of tumor instability are PCR-based. PCR-based methods may underestimate mutation frequencies of heterogeneous tumor genomes. Using a novel PCR-free random cloning/sequencing method, we analyzed 100 kb of total genomic DNA from blood lymphocytes, normal prostate and tumor prostate taken from six individuals. Variations were identified by comparison of the sequence of the cloned fragments with the nr-database in Genbank. After excluding known polymorphisms (by comparison to the NCBI dbSNP), we report a significant over-representation of variants in the tumors: 0.66 variations per kilobase of sequence, compared with the corresponding normal prostates (0.14 variations/kb) or blood (0.09 variations/kb). Extrapolating the observed difference between tumor and normal prostate DNA, we estimate 1.8 million somatic (de novo) alterations per tumor cell genome, a much higher frequency than previous measurements obtained by mostly PCR-based methods in other tumor types. Moreover, unlike the normal prostate and blood, most of the tumor variations occur in a specific motif (P = 0.046), suggesting common etiology. We further report high tumor cell-to-cell heterogeneity. These data have important implications for selecting appropriate technologies for cancer genome projects as well as for understanding prostate cancer progression.


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