Methods for Improving Challenging DNA Profiles and Molecular Preservation of Soft Tissue Samples

Abstract

Degradation of DNA can lead to either poor quality, imbalanced, or even no profiles. Therefore, appropriate collection and storage methods are critical to minimize its impact. If the DNA is degraded prior to sample collection, then the degradation process can only be arrested and other methods have to be employed to try to improve the quality of the DNA profile. The major aims of this thesis were to assess alternative methods for molecular preservation of muscle tissue samples and to obtain better DNA profiles from degraded samples.
Assessment of DNA degradation was undertaken using an in-house PCR assay which amplifies four amplicons from 70 bp to 384 bp. DNA degradation was evaluated in whole pig carcasses exposed to hot and humid environmental conditions. A full DNA profile could be generated for 24 hours, but some full profiles were obtained from samples taken as late as 72 hours. It was determined that when collecting tissue samples from partially decomposed bodies, those should be preferentially from the surface of the body in touch with the ground, as the results show that DNA persistence is improved.
In order to compare field and laboratory degradation patterns, muscle tissue samples were incubated in the laboratory at 25 °C and 37 °C. The persistence of DNA was increased when compared to field, most likely due to the lack of insect activity and of variations in temperature and humidity. Partially degraded muscle samples were preserved with 96% ethanol, cell lysis solution, or cell lysis solution with 1% sodium azide, which had been stored at room temperature for seven years. Samples were re-extracted to assess the long-term efficacy of these storage solutions. The results show that ethanol and cell lysis solution with 1% sodium azide were successful in preserving DNA for this period.
Fresh muscle tissue samples were stored at 25 °C and 37 °C for up to 42 days using vodka and 37.5% ethanol as preservatives. Complete amplification profiles were obtained up to the last time point from samples that had any preservative solution, while samples left untreated had dropouts after 14 days. It is recommended that the use of drinking ethanol should be considered in situations where the stock of absolute ethanol is limited. The possibility of using vacuum for preservation was tested on fresh muscle tissue samples incubated at 25 °C and 37 °C. The results show that even if there was a limited amount of air inside the storage bag, and not complete vacuum, DNA persistence was enhanced when compared to samples incubated at the same conditions in plastic tubes.
Some approaches were attempted to improve degraded DNA profiles. First, degraded DNA was selectively extracted from agarose gels to manipulate the proportion of longer and smaller DNA fragments present. Despite promising preliminary results, this technique showed no usefulness in improving DNA profiles. Purification columns were used with the same aim, but when comparing the original sample with the processed samples, the best results obtained were of equivalence. As an alternative approach, a protocol of DNA Capture was developed in an attempt to preferentially extract the fragments to be analysed in a degraded DNA sample in equal amounts. Whilst the DNA capture method worked in preliminary experiments, it was not applied to degraded profiles.
The results obtained have allowed recommendations around collection (i.e. how long samples could be viable for DNA analysis) and storage to be refined. Attempts to rebalance already degraded profiles were not successful. Future field experiments planned as a follow up to the work presented involve testing collection methods and the effectiveness of vacuum body bags.