Date of Submission
Angie Ambers, Ph.D.
Postmortem Survival of DNA, Human Skeletal Remains, PCR Inhibitors, Quantitative PCR
Humic Substances, Forensic Genetics, Body Remains, Polymerase Chain Reaction, DNA Fingerprinting
Humic acid, Forensic genetics, Dead, Anthropometry, Polymerase chain reaction, DNA fingerprinting—Technique
Postmortem survival of DNA in human skeletal remains occurs due to the compact microstructure of the skeleton and its ability to provide a strong, protective physical barrier to environmental insults. On a molecular level, DNA preservation in bones/teeth involves electrostatic interactions between the negatively-charged DNA backbone and positively charged calcium residues in hydroxyapatite, the latter of which is one of the main components of bone microstructure. Despite these protections, over time endogenous DNA becomes damaged, limiting our ability to detect it and affecting its utility in making a positive identification. Hence, forensic genetic investigations of unidentified human remains (UHRs) are limited by the quality, quantity, and purity of DNA recovered.
Significant damage or alteration to the molecular structure of DNA is problematic because polymerases stall at damaged/altered sites, preventing PCR amplification (and subsequent analysis) of target loci. Concurrent complications arise from endogenous and/or environmental inhibitors that tend to co-extract with DNA and impede or completely block downstream polymerase-based reactions. One of the most pervasive PCR inhibitors encountered in skeletal remains cases is humic acid (HA), an acid found in all soils worldwide, in varying concentrations. Purification of endogenous DNA away from such an inhibitor is crucial for both the quantification and PCR amplification steps in the forensic DNA workflow.
The purpose of this study was to demonstrate the effect of co-extracted humic acid (HA) on quantitative PCR (qPCR), the method used to determine the amount of DNA recovered from evidentiary samples. The inhibitory effects of six different HA solutions (5.0mg/mL, 2.5mg/mL, 1.25mg/mL, 0.625mg/mL, 0.3125mg/mL, 0.156mg/mL) on six different DNA concentrations (50ng/µL, 5ng/µL, 0.5ng/µL, 0.05ng/µL, 0.02ng/µL, 0.005ng/µL) were explored. At the three highest HA concentrations (5.0mg/mL, 2.5mg/mL, 1.25mg/mL), complete qPCR inhibition was observed for all DNA concentrations. At the lowest three HA concentrations (0.625mg/mL, 0.3125mg/mL, 0.156mg/mL), DNA polymerases in the qPCR assay were able to work, but with lower efficiency. Even in the presence of these low HA concentrations, accuracy of DNA quantification was reduced (i.e., the qPCR assay under-estimated DNA quantities present for all samples). This under-estimation could substantially impact downstream PCR amplification of STR loci and may result in partial DNA profiles or no DNA profiles. Purification of DNA from the bone matrix is essential. The results of this study demonstrate the importance of effective DNA extraction and removal of inhibitors to maximize chances of DNA typing success.
King, Madeline, "Evaluation of the Effect of the Humic Acid Inhibitor on Forensic Genetic Investigations of Human Skeletal Remains" (2021). Honors Theses. 13.