Published online 1 October 2009 Nucleic Acids Research, 2009, Vol. 37, No. 22 7441–7446 doi:10.1093/nar/gkp761 PCR-free method detects high frequency of genomic instability in prostate cancer Nick M. Makridakis1,*, Troy Phipps 2 , 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 Received May 5, 2009; Revised August 28, 2009; Accepted August 31, 2009 ABSTRACT 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. INTRODUCTION Prostate cancer is the most common malignancy in men, with over 300 000 diagnoses and 30 000 deaths in the United States alone (1). Underlying factors implicated in disease etiology include diet, ethnicity, genetic predisposi- tion, hormones and other environmental contributors (2). Most experts agree that genomic instability plays a role during cancer evolution, but its exact signiﬁcance is debated (3). Two types of genomic instability are evident in most cancers (3): large-scale alterations (e.g. aneuploidy, translocations) and small-scale alterations (e.g. single nucleotide substitutions, microsatellite instabil- ity). The extent of small-scale somatic alterations has been mostly probed with PCR-based methods: the majority of tumors display low frequencies of genomic instability (4–6) although a screen of the complete protein kinase gene family (which contains genes that are commonly mutated in tumors) revealed the equivalent of 142 000 alterations per tumor cell (7). PCR-based methods however are bound to underestimate the total number of variations present in heterogeneous genomes because they only record the most common genotypes in a pool of het- erogeneous molecules (8). In order to account for tumor heterogeneity one needs to employ cell-speciﬁc or single molecule analyses, such as sequencing individual clones. Single molecule PCR analysis has resulted in the discov- ery of signiﬁcant instability in various types of tumors (9), but the target sequence analyzed is a 4-bp fragment in a p53 intron, making it diﬃcult to extrapolate those mutation frequencies for the whole cancer genome. Single molecule methods based on next generation sequencing have identiﬁed somatic tumor mutations not detected by Sanger sequencing (10), but the cost per sequencing run is signiﬁcant, and the relatively high back- ground makes it impossible to identify mutations present in <0.2% of the tumor genome (10). Furthermore, both of the above methods are based on the presumed reduction of heterogeneous tumor samples to single molecules by dilution (before ampliﬁcation). It is technically much harder to guarantee generation of single DNA molecules by dilution, compared to, e.g. cloning. Accurate measure- ment of the extent of genomic instability in tumors is important for understanding cancer etiology, disease prognosis and for successful treatment. We recently reported a high frequency of somatic mutations in several genes in prostate cancer tissue, and *To whom correspondence should be addressed. Tel: 504-9885418; Fax: 504-9885516; Email: nmakrida@tulane.edu The Author(s) 2009. 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