Methodology article

Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues

Sarit Aviel-Ronen^1,2 , Chang Qi Zhu² , Bradley P Coe³ , Ni Liu² , Spencer K Watson³ , Wan L Lam³ and Ming Sound Tsao^1,2,4

¹  Department of Pathology, University Health Network, 200 Elisabeth Street, Toronto, ON M5G 2C4, Canada

²  Division of Applied Molecular Oncology, Ontario Cancer Institute/Princess Margaret Hospital, University Health Network, 610 University Avenue, Toronto, ON M5G 2M9, Canada

³  Department of Cancer Genetics and Developmental Biology, British Columbia Cancer Research Centre, 675 West 10^th Avenue, Vancouver, BC V5Z 1L3, Canada

⁴  Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto, 100 College Street, Toronto ON M5G 1L5, Canada

author email corresponding author email

BMC Genomics 2006, 7:312doi:10.1186/1471-2164-7-312

Published:	12 December 2006

Abstract

Background

Formalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer. Therefore, it is desirable to develop methods that enable whole genome amplification (WGA) using DNA extracted from FFPE tissues. Multiple-strand Displacement Amplification (MDA) is an isothermal method for WGA that uses the large fragment of Bst DNA polymerase. To date, MDA has been feasible only for genomic DNA isolated from fresh or snap-frozen tissue, and yields a representational distortion of less than threefold.

Results

We amplified genomic DNA of five FFPE samples of normal human lung tissue with the large fragment of Bst DNA polymerase. Using quantitative PCR, the copy number of 7 genes was evaluated in both amplified and original DNA samples. Four neuroblastoma xenograft samples derived from cell lines with known N-myc gene copy number were also evaluated, as were 7 samples of non-small cell lung cancer (NSCLC) tumors with known Skp2 gene amplification. In addition, we compared the array comparative genomic hybridization (CGH)-based genome profiles of two NSCLC samples before and after Bst MDA. A median 990-fold amplification of DNA was achieved. The DNA amplification products had a very high molecular weight (> 23 Kb). When the gene content of the amplified samples was compared to that of the original samples, the representational distortion was limited to threefold. Array CGH genome profiles of amplified and non-amplified FFPE DNA were similar.

Conclusion

Large fragment Bst DNA polymerase is suitable for WGA of DNA extracted from FFPE tissues, with an expected maximal representational distortion of threefold. Amplified DNA may be used for the detection of gene copy number changes by quantitative realtime PCR and genome profiling by array CGH.