Validation of whole genome amplification for analysis of the p53 tumor suppressor gene in limited amounts of tumor samples

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• 作者：Johanna Hasmats ; Henrik Green ; Beata Werne Solnestam ; Pawel Zajac ; Mikael Huss ; Cedric Orear ; Pierre Validire ; Magnus Bjursell ; Joakim Lundeberg
• 关键词：Whole genome amplification ; TP53 ; Mutations ; Validation
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2012
• 出版时间：24 August, 2012
• 年：2012
• 卷：425
• 期：2
• 页码：379-383
• 全文大小：455 K

文摘

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 ¦Õ29 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.