The archives of formalin-fixed, paraffin-embedded (FFPE) tissue sections represent a valuable and extensive source of material for biomedical research, however, DNA derived from FFPE samples is usuallyl highly degraded and cross-linked due to fixation. This section describes innovative REPLI-g technology that enables the successful amplification of fragmented and FFPE DNA samples.
Working with fragmented DNA and fixed tissue samples
Multiple Displacement Amplification requires average genomic DNA fragment sizes of approximately 2 kb in order to amplify DNA without introducing any bias. Fragmented or low-quality DNA can be used as long some DNA fragments are above 2 kb in length. This is because randomly fragmented DNA should contain multiple intact copes of each locus.
DNA derived from formalin-fixed, paraffin-embedded (FFPE) tissue samples is usually highly degraded and cross-linked, due to fixation. Formalin fixation is a commonly used technique for preserving tissue samples for paraffin embedding. The fixation ensures the preservation of tissue architecture and cell morphology by cross-linking biomolecules. Different sample types may require a different fixation procedure: tissues with a soft consistency, such as breast tissue samples, usually require a longer fixation step to preserve tissue morphology (see figure Effect of tissue fixation on DNA
). Longer fixation times may result in two effects: a higher degree of cross-links is generated between biomolecules, and a higher degree of DNA fragmentation occurs — resulting in small DNA fragments of usually several hundred base pairs in length.
DNA obtained from FFPE tissue samples is not long enough to be amplified successfully by standard MDA procedures. The REPLI-g FFPE Kit uses an integrated processing reaction that results in isolation of DNA from FFPE samples and ligation of DNA fragments in a random order (see figures REPLI-g FFPE Procedure
and High-molecular-weight ligated DNA
). The resulting DNA is then amplified using REPLI-g Polymerase, as shown in the figure Random DNA ligation
and can be successfully used for sensitive downstream applications (see figure Reliable microsatellite analysis