Abstract
Since the completion of the Human Genome Project in 2003, the nucleic acid (NA) code has continually been leveraged to provide actionable information to patients for personalized care and to enable statistically irrefutable matches for forensic human identification (HID). As such, the research and development focused on the miniaturization and automation of tools for NA analysis has increased year after year. Regardless of the application, the methods associated with the preparation of genetic material for downstream analysis are arguably the most vital. Briefly, the ability to recover and conserve NAs from sample enrichment, DNA extraction, or epigenetic conversion will ultimately be outcome determinative of any associated assay. And yet, many of these aforementioned benchtop techniques are considered labor-intensive, time-consuming, and difficult to automate due to the harsh associated reagents, lengthy incubations, and multitude of required pipetting steps. Furthermore, the performance of each process is inconsistent from analyst-to-analyst and resulting in variable NA loss. This body of work describes multiple microfluidic approaches aimed to streamline and enhance genetic and epigenetic sample preparation methods, including bisulfite conversion, DNA extraction, and virion enrichment.
As an alternative to forensic genetic identification by short tandem repeats (STRs), probing epigenetic variation can provide information related to HID. In particular, examining the methylation of cytosine at targeted locations along the genome has provided mechanisms to differentiate between monozygotic twins, predict smoking habits, and estimate chronological age within ~1 year of accuracy, to name a few. Unlike conventional testing by STR analysis, methylation interrogation requires an extensive, multi-step sample preparation process resulting in a magnitude of DNA loss. In particular, the bisulfite conversion (BSC) technique, which chemically modifies all cytosine residues not containing a methyl tag to uracil, is known to result in the loss of more than 50% of DNA. Chapter 2 describes the development of a rotationally-driven microfluidic method for dynamic solid phase-BSC (dSP-BSC) that automates the sample preparation process for up to four samples in parallel. By leveraging the microfluidic format and reduced reaction volumes, incubation intervals were shortened by ~40% overall with maintained DNA recovery and conversion efficiency compared to the conventional approach.
Chapter 3 focuses on the integration of DNA extraction by enzymatic lysis upstream from BSC to enhance DNA recovery and couple the sample preparation workflow from extraction to conversion. While gold-standard methods for DNA extraction involve cellular lysis followed by silica-assisted purification of NAs, this work incorporates an alternative method for DNA extraction based upon the introduction of a highly thermostable neutral protease from Bacillus sp. strain EA1. Extraction by the EA1 enzyme permits NA preparation in a single tube and eliminates the need for pro K, SDS, harsh chemicals, and silica purification altogether. Herein, compatibility between EA1 and downstream BSC is established for the first time. Performance is evaluated with standards, K-562 samples, and blood samples. Toward a fully integrated approach to epigenetic sample preparation, a rotationally-driven device design in proposed and assessed using colorimetric dye studies and preliminary testing with venous blood samples.
Utilizing similar microfluidic design features as those proposed in Chapters 2 – 3, Chapter 4 presents a method for the sample preparation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) aimed for diagnostic testing via downstream RT-PCR. This work was completed in response to the 2020 global pandemic and with support from the National Institutes of Health (NIH) Rapid Acceleration of Diagnostics (RADx) initiative to enhance viral surveillance and containment efforts. Once again, two sample preparation processes were coupled via a rotationally-driven microfluidic interface. These included the enrichment of SARS-CoV-2 virions by affinity-capture, magnetic nanoparticles and extraction by the EA1 enzyme. With the integrated device, sample preparation was possible in under 15 minutes and for a total of up to six samples in parallel. Performance of the device was comparable to a gold-standard method for sample preparation and evaluated using standards, patient nasopharyngeal swabs, and contrived saliva samples.
Chapter 5 highlights the ongoing studies and future work toward the microfluidic integration of epigenetic sample preparation from extraction to conversion and discusses persistent challenges associated with pyrosequencing and material-related fluid loss and inhibition. Considering much of the work detailed in this dissertation was focused on the development of a microfluidic tool for forensic genetic integration, the remaining focus of Chapter 5 highlights multiple trade-offs faced in the forensic landscape as it relates to the adaptation of new scientific methods. Opportunity zones for microfluidic research and development within the bounds of HID and criminal investigation are posited that might increase laboratory efficiency and expand the capacity of forensic science services in the future.
