title: Validation of whole genome amplification for analysis of the p53 tumor suppressor gene in limited amounts of tumor samples.
abstract: Personalized cancer treatment requires molecular characterization of individual tumor biopsies. These samples are frequently only available in limited quantities hampering genomic analysis. Several whole genome amplification (WGA) protocols have been developed with reported varying representation of genomic regions post amplification. In this study we investigate region dropout using a φ29 polymerase based WGA approach. DNA from 123 lung cancers specimens and corresponding normal tissue were used and evaluated by Sanger sequencing of the p53 exons 5-8. To enable comparative analysis of this scarce material, WGA samples were compared with unamplified material using a pooling strategy of the 123 samples. In addition, a more detailed analysis of exon 7 amplicons were performed followed by extensive cloning and Sanger sequencing. Interestingly, by comparing data from the pooled samples to the individually sequenced exon 7, we demonstrate that mutations are more easily recovered from WGA pools and this was also supported by simulations of different sequencing coverage. Overall this data indicate a limited random loss of genomic regions supporting the use of whole genome amplification for genomic analysis.
authors: Science for Life Laboratory, School of Biotechnology, Division of Gene Technology, Royal Institute of Technology, Stockholm, Sweden.
authors: Johanna
authors: J
authors: Hasmats
authors: Henrik
authors: H
authors: Green
authors: Beata Werne
authors: BW
authors: Solnestam
authors: Pawel
authors: P
authors: Zajac
authors: Mikael
authors: M
authors: Huss
authors: Cedric
authors: C
authors: Orear
authors: Pierre
authors: P
authors: Validire
authors: Magnus
authors: M
authors: Bjursell
authors: Joakim
authors: J
authors: Lundeberg
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000032:analysis* / Q000096:biosynthesis; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D005091:Exons; D015894:Genome, Human; D023281:Genomics / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
chemical_list: D004273:DNA, Neoplasm; D016159:Tumor Suppressor Protein p53; D004259:DNA-Directed DNA Polymerase