Abstract
BackgroundFormalin-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.ResultsWe 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.ConclusionLarge 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.
Highlights
Formalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer
We have investigated the feasibility of Multiple-strand Displacement Amplification (MDA) on DNA from FFPE tissue using the Bst DNA polymerase, and evaluated the magnitude of representational distortion using quantitative realtime polymerase chain reaction (PCR) (QPCR) and whole genome tiling array comparative genomic hybridization (CGH) representing complete coverage of the human genome
All tested genes were found in all FFPE and Bst amplified samples, and their relative gene copy number was within 3-fold range of Isothermal amplification methods refer to Hyperbranched Strand Displacement Amplification (HSDA), which is known as Multiple-strand Displacement Amplification (MDA) [11,12,13]
Summary
Formalin-fixed paraffin-embedded (FFPE) tissues represent the largest source of archival biological material available for genomic studies of human cancer. 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. It is desirable to establish a whole genome amplification (WGA) method for DNA extracted from FFPE tissues. What common in all these protocols are the PCR principle of temperature-dependent cyclic amplification, and the use of primers with a random sequence to allow for multiple binding sites. They differ in primer design and the sequence of temperature changes. Each technique has its advantages and limitations, varying from incomplete genomic coverage to preferences for certain DNA length (e.g. shorter alleles in DOP-PCR [4]), and inconsistency in the magnitude of amplification and elaborated protocol (SCOMP)
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