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Genomic Analysis of Formalin-Fixed Paraffin Embedded (FFPE) Tissues through the use of Whole Genome Amplification (WGA)

he method has been successfully applied to formalin-fixed samples.16 There has been some confusion as to the mechanism of this system, which was first described in a 2004 Sigma-Aldrich Life Science Quarterly article.17 GenomePlex uses a mild fragmentation step to randomly break up long DNA, which facilitates subsequent steps. Priming is accomplished using an oligo with known 5 ends but degenerate 3- ends, and the multicopy DNA sample is reproduced in a series of overlapping single stranded DNAs. These single stranded products are subsequently primed with the same oligo to create double stranded DNA with fixed 5- ends. The resulting library is amplified using PCR, Figure 1 .



The GenomePlex hybrid method has several advantages over the previously described WGA methods. It does not require template with long, amplifiable regions, as does MSD. The method does not rely on highly efficient digestion and ligation as does LAP amplification. Finally, GenomePlex has improved on PEP and DOP amplification with the design of improved primers, and does not suffer from reduced amplification due to competing primer interactions.

There are limits to all WGA techniques, and tissue fixation by formaldehyde is a highly varied procedure that affords extracted DNA of vastly different qualities. As related above, MSD requires relatively long stretches of amplifiable DNA to create a representative product. LAP, in contrast, relies on both a highly efficient restriction digest and complete ligation in order to deliver representative amplification. In contrast, GenomePlex primers and cycling parameters have been optimized to prime, on average, twice for every ~1 kb stretch of genomic DNA. Therefore, when starting with genomic DNA fragments that have been severely degraded (below 1 kb) GenomePlex will amplify at less than optimal efficie
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