DNA degradation in post-mortem soft muscle tissues in relation to accumulated degree-days (ADD)

Abstract

In order to assess DNA degradation in the model organisms chosen (pig and rabbit), two nuclear genes, Connexin 43 and RAG-1, were aligned to identify conserved regions. Primers were designed to amplify 70 bp, 194 bp, 305 bp and 384 bp amplicons. The primers were also designed to amplify human DNA, which allowed the use of commercially purchased DNA standards to be used as controls. Following DNA extraction PCR analysis was performed using the four primers sets in a multiplex (4-plex): the PCR was optimised so that it worked over a wide range of template amounts (0.1–75.83 ng). The multiplex (4-plex) PCR was found to work efficiently in triplicate samples with all three species down to 0.3 ng of DNA template. This multiplex has been used to assess whether DNA degradation can be predicted by accumulated degree-days (ADD), which provides a measure of both time and temperature. Full 4-plex profiles were generated until day 7 (112 ADD) from whole carcasses and body fragments. Future work will include; development of real-time PCR quantification assays, DNA fragment analysis and DNA preservation.