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

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.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.With growing interest in the genomic characteristics of various human tumors and a steep increase in the availability of genomic tests for both clinical and research purposes, the amount of genomic DNA available from biological samples may limit the practicality of genomic analysis. Having been used for decades, formalin-fixed paraffin-embedded (FFPE) tissues comprise the most common form of human tissue samples archives. Therefore, it is desirable to establish a whole genome amplification (WGA) method specifically for DNA extracted from FFPE tissues. Two main approaches for WGA have been developed: thermocycling protocols and isothermal amplification methods.Several thermocycling protocols have been used, including the degenerate oligonucleotide prim