Formalin fixation and paraffin embedding cause DNA fragmentation. During the REPLI-g FFPE procedure, these DNA fragments are randomly ligated and then amplified using multiple displacement amplification. As indicated, DNA fragments (containing different loci [L1–L4]) are not assembled in the original order. However, the different loci are equally amplified and can be detected in downstream genotyping applications.
DNA from different FFPE carcinoma tissues after (1a–5a) and before (1b–5b) amplification using the REPLI-g FFPE Kit was run on an agarose gel. The REPLI-g FFPE ligation procedure results in the formation of high-molecular-weight DNA. (Data kindly provided by Hartmut Schmidt, Gerhard Domagk Institute for Pathology, Münster, Germany.)
DNA from a 3.5-year-old FFPE carcinoma tissue sample was analyzed at a single microsatellite locus prior to and after DNA amplification using the REPLI-g FFPE Kit. Identical results were obtained for the original and REPLI-g amplified genomic DNA. (Data kindly provided by Hartmut Schmidt, Gerhard Domagk Institute for Pathology, Münster, Germany.)
The REPLI-g FFPE procedure involves a novel DNA processing reaction that prepares and ligates fragmented DNA. This ligated DNA is then used in MDA-based whole genome amplification.