Whole genome amplification from challenging sample types
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.
Effect of tissue fixation on DNA. DNA fragmentation reduces the sensitivity of downstream assays as the locus of interest may not be amplified.
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).
REPLI-g FFPE Procedure. 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.
High-molecular-weight ligated DNA. 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 provided by Hartmut Schmidt, Gerhard Domagk Institute for Pathology, Münster, Germany.)
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).
Random DNA ligation. The REPLI-g FFPE procedure involves a novel DNA processing reaction which prepares and ligates fragmented DNA. Whole genome amplification of this randomly ligated DNA is then performed using proven REPLI-g technology. REPLI-g products combine MDA with a uniquely processive DNA polymerase. Due to the high processivity and strand displacement activity, REPLI-g DNA Polymerase minimizes unequal sequence and locus representation. This provides much more reliable results when compared to PCR-based WGA methods
Reliable microsatellite analysis. 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 provided by Hartmut Schmidt, Gerhard Domagk Institute for Pathology, Münster, Germany.)