DNA Persistence and Preservation Following Environmental Insult

Nazir, Muhammad Shahid (2012) DNA Persistence and Preservation Following Environmental Insult. Doctoral thesis, University of Central Lancashire.

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This research was conducted to provide empirical evidence to supplement advice available to the forensic community for the collection of muscle tissue for forensic analysis. This type of collection is normally carried out to determine the identity of individuals following mass disasters, such as plane crashes or natural disasters.

DNA degradation was assessed in two model organisms, pig and rabbit (with human DNA as a control), over various time points. Rabbit recombination activating gene (RAG 1) was aligned to identify conserved regions in pig, rabbit and human. Primers were designed and optimised to create a 4-plex PCR multiplex that can amplify 70 bp, 194 bp, 305 bp and 384 bp in three species. The 4-plex multiplex was found to work efficiently in all three species down to 0.3 ng of DNA template. The multiplex was used to assess whether DNA degradation can be predicted by accumulated degree-days (ADD), which provides a measure of both time and temperature. A series of field studies were performed to assess DNA persistence in pig and rabbit soft muscle tissues using a combination of whole animals, suspended muscle tissues (insect activity free) and muscle fragments. Field studies were carried out in: August-September 2009; February-May 2010; May-June 2010; June-July 2010 and September-November 2010. Soft muscle tissue samples were collected at different ADD.

4-plex multiplex results showed that DNA was more persistent in pig tissues compared to rabbit tissues. In the September 2010 experiments, full multiplex amplification was obtained from rabbit until 137 ADD (whole carcases) and 210 ADD (body fragments and suspended tissues), while in the August 2009 experiments, full multiplex amplification was obtained until 112 ADD (whole carcases and body fragments) and until 141 ADD (suspended tissues). In the June 2010 experiments, full multiplex amplification was possible until 64 ADD.

Pig whole carcases which were placed in the field in February 2010, showed multiplex amplification until day 90 (603 ADD), followed by September 2010 (until day 44 (490 ADD)) and May 2010 (until day 27 (338 ADD)). During the September 2010 project, body fragments produced full amplification until muscles were collected (342 ADD), while in case of whole carcases and suspended tissues; the amplification was possible until 490 ADD. There was complete failure of amplification of 305 bp and 384 bp in pig whole carcases after 342 ADD, while in suspended tissues, the amplification of 305 bp and 384 bp was possible until 420 ADD.

The statistical analysis showed that amplification success of larger amplicons (194 bp, 305 bp and 384 bp) reduces with increase in ADD in pig and rabbit whole carcases, body fragments and suspended tissues while 70 bp was more persistence. The results showed that there was no significant difference in DNA persistence between whole carcases verses suspended tissues (Z=0.57, p>0.05) and whole carcases verses body fragments (Z=1.71, p>0.05), There was however a significant difference (Z=2.31, p<0.05) in DNA persistence in suspended tissues and body fragments with increase in ADD. The results from field experiments suggested that muscle tissues, if available, should be collected for DNA profiling, since even if degraded, a profile can be obtained. The results also suggested that the isolation of tissues from insect activity as quickly as possible (even if immediate storage is not possible) may be beneficial for DNA persistence. Seasonal variation in DNA persistence was observed due to maggot mass growth which increases carcase decomposition and ultimately effect on DNA persistence.

Controlled incubation experiments were also performed at 27 °C, 37 °C and 47 °C until 21 days to assess DNA persistence, as these temperatures were not available under field conditions. The results showed that the amplification of 70 bp was more persistent compared to larger amplicons (194 bp, 305 bp and 384 bp). The drop-out in amplification of larger amplicons occurred more rapidly in samples incubated under laboratory conditions compared to the field samples. The statistical analysis showed species, ADD and temperature have strong effect (p<0.05) on DNA persistence under controlled conditions.

The appearance of 70 bp amplicons in all samples collected from field and in most samples from controlled incubation experiments suggested that soft muscle tissues exposed to different environments can be used to perform SNP analysis.
The full 4-plex multiplex amplification obtained from rabbit and pig preserved and dehydrated samples suggested that 96% ethanol, cell lysis solution (with and without 1% sodium azide) and dehydration can be used to preserve fresh and partially decomposed soft muscle tissues at room temperature for one year. The drop-out in amplification of larger amplicons in tissues preserved in 10% buffered formalin suggested that formalin was not suitable for long term storage. This system should therefore be considered as an additional method during Disaster victim identification (DVI) work to preserve fresh and partially decomposed samples. This study also suggested that the developed multiplex (4-plex) can be used to assess DNA persistence in human decomposing bodies and in experimental studies.

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