Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor's Name

Bruce McCord

First Advisor's Committee Title

Committee Chair

Second Advisor's Name

DeEtta Mills

Second Advisor's Committee Title

Committee Member

Third Advisor's Name

Alexander Mebel

Third Advisor's Committee Title

Committee Member

Fourth Advisor's Name

George Duncan

Fourth Advisor's Committee Title

Committee Member

Fifth Advisor's Name

Kathleen Rein

Fifth Advisor's Committee Title

Committee Member

Keywords

DNA Inhibitors, Direct Analysis in Real-Time (DART), Real-Time PCR, DNA purification, Metal-DNA complex (M-DNA), clandestine grave sites, soil microbial metagenomics

Date of Defense

3-23-2015

Abstract

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.

Identifier

FI15032192

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