Characterizing variability in fluorescence-based forensic DNA measurement and developing an electrochemical-based quantification system
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A reliable and robust laboratory method is essential for the forensic analysis of deoxyribonucleic acid (DNA), particularly for low-template samples. Electropherogram peak heights are important to the identification of STR alleles, and these peak heights are prone to error. Since error can be introduced into the process during sample preparation, quantification, amplification, or analysis, validation studies are performed in an attempt to characterize the signal variation associated with the process. While current practices assess aspects of a method, such as sensitivity and reproducibility, the effects of daily laboratory alterations are often not considered. Additionally, samples used in a validation study may be prepared using serial dilutions. Therefore, understanding the extent to which error is propagated through the series and the effect it has on the results could help improve validation practices. This work aimed to assess the effect daily laboratory modifications have on the signal in a forensic electropherogram. Specifically, the variability in signal when different capillary and amplification kit lots were used was evaluated against the variability observed when a single sample was either injected or amplified multiple times. The variability was determined via the examination of peak heights, peak height ratios, stutter, and drop-out. The effect of serially diluting samples was examined via an in silico model of the dilution process, polymerase chain reaction (PCR), and capillary injection. The peak heights from simulated serially diluted samples using the concentration of a stock DNA were compared to the peak heights from simulated samples that were quantified after the dilution series was generated and prior to amplification. The different capillary lots and amplifications were found to result in greater variation compared to the multiple injections. Additionally, when the stutter percentages obtained from using multiple kit lots were compared to those obtained using the same kit lot, differences in stutter percentage deviations resulted in different stutter thresholds. Drop-out rates were also different between the samples amplified with one kit versus the same samples amplified with multiple kit lots. Therefore, at a minimum, multiple amplifications should be run on multiple capillary lots during validation. Further, if available, the use of multiple kit lots is recommended, particularly in cases where stutter thresholds or drop-out models are used during interpretation. Creating validation samples via serial dilutions was also found to increase the variation observed in peak height in the simulated samples, suggesting that samples should be quantified post-dilution. In addition to characterizing the variability of several components of DNA analysis, an alternative quantification method was investigated in order to decrease the overall variability associated with the quantification process. This work sought to develop an electrochemical biosensor using a single-stranded DNA (ssDNA) probe chemically adsorbed to a gold electrode. This would allow for the direct quantification of DNA and eliminate the need for qPCR and fluorescent-based oligonucleotide detection systems. The DNA probe was successfully adsorbed to the surface of the gold disk electrode, hybridized to a single-stranded complementary DNA sequence, and detected using square wave voltammetry. Additionally, the ability to control the amount of DNA chemisorbed to the electrode surface was investigated by varying the incubation time in the probe solution. The measured current increased as the incubation time increased from 15 minutes to one hour, after which it plateaued. The use of an electrochemical biosensor is a promising alternative to qPCR for the quantification of DNA, with one hour being the optimal incubation time in the probe solution.