Optimized manual and automated recovery of amplifiable DNA from tissues preserved in buffered formalin and alcohol-based fixative.
ABSTRACT Archival tissue preserved in fixative constitutes an invaluable resource for histological examination, molecular diagnostic procedures and for DNA typing analysis in forensic investigations. However, available material is often limited in size and quantity. Moreover, recovery of DNA is often severely compromised by the presence of covalent DNA-protein cross-links generated by formalin, the most prevalent fixative. We describe the evaluation of buffer formulations, sample lysis regimens and DNA recovery strategies and define optimized manual and automated procedures for the extraction of high quality DNA suitable for molecular diagnostics and genotyping. Using a 3-step enzymatic digestion protocol carried out in the absence of dithiothreitol, we demonstrate that DNA can be efficiently released from cells or tissues preserved in buffered formalin or the alcohol-based fixative GenoFix. This preparatory procedure can then be integrated to traditional phenol/chloroform extraction, a modified manual DNA IQ or automated DNA IQ/Te-Shake-based extraction in order to recover DNA for downstream applications. Quantitative recovery of high quality DNA was best achieved from specimens archived in GenoFix and extracted using magnetic bead capture.
Article: Long-term room temperature preservation of corpse soft tissue: an approach for tissue sample storage.[show abstract] [hide abstract]
ABSTRACT: Disaster victim identification (DVI) represents one of the most difficult challenges in forensic sciences, and subsequent DNA typing is essential. Collected samples for DNA-based human identification are usually stored at low temperature to halt the degradation processes of human remains. We have developed a simple and reliable procedure for soft tissue storage and preservation for DNA extraction. It ensures high quality DNA suitable for PCR-based DNA typing after at least 1 year of room temperature storage. Fragments of human psoas muscle were exposed to three different environmental conditions for diverse time periods at room temperature. Storage conditions included: (a) a preserving medium consisting of solid sodium chloride (salt), (b) no additional substances and (c) garden soil. DNA was extracted with proteinase K/SDS followed by organic solvent treatment and concentration by centrifugal filter devices. Quantification was carried out by real-time PCR using commercial kits. Short tandem repeat (STR) typing profiles were analysed with 'expert software'. DNA quantities recovered from samples stored in salt were similar up to the complete storage time and underscored the effectiveness of the preservation method. It was possible to reliably and accurately type different genetic systems including autosomal STRs and mitochondrial and Y-chromosome haplogroups. Autosomal STR typing quality was evaluated by expert software, denoting high quality profiles from DNA samples obtained from corpse tissue stored in salt for up to 365 days. The procedure proposed herein is a cost efficient alternative for storage of human remains in challenging environmental areas, such as mass disaster locations, mass graves and exhumations. This technique should be considered as an additional method for sample storage when preservation of DNA integrity is required for PCR-based DNA typing.Investigative genetics. 08/2011; 2:17.