In forensic science, it is important to have the capability to collect, stabilize and store deoxyribonucleic acid (DNA) in an efficient manner so that successful and probative DNA profiles can be generated. The success of generating downstream profiles is dependent on viable DNA extracts, thus the preservation and protection of the samples from degradation or loss is imperative. According to the National Institute for Standards and Technology (NIST) Biological Evidence Preservation Handbook, dried biologically stained samples are best stored in a temperature controlled environment, which is defined as less than 60% humidity with a temperature maintained between 15.5°C and 24°C. Presently, long-term forensic evidence samples, in the form of DNA extracts, have a preservative added and are either refrigerated (2°C – 8°C) or stored in freezers with temperatures typically set at or below -10°C[1]. However, using freezers as a means of sample storage can result in limited storage space, high cost, and risk of electrical failure[2].
Alternative storage methods that can be employed in ambient conditions have been implemented in some forensic laboratories and are suitable to protect and preserve DNA evidence. Different stabilizing agents such as SampleMatrix™ and DNAstable™ have been studied to assess their ability at storing and stabilizing extracted DNA at ambient temperatures. Research by Lee et al., showed DNA stored in SampleMatrix™ was protected against degradation for up to 12 months and maintained its integrity, resulting in a 2-fold increase of recoverability over samples stored in a freezer for the same amount of time. This study also showed that DNA stored in SampleMatrix™ did not affect the PCR amplification process and samples that were desiccated and stored in SampleMatrix™ resulted in similar completeness of STR profiles with samples stored at -20°C for 0.125 ng and 0.25 ng DNA concentrations over the course of 12 months[3]. DNAstable™ was also shown to have similar storage and stabilizing capabilities over a long period of time under various temperature conditions.
Mawi DNA Technologies’ iSWAB™-ID collection system and its associated buffer is capable of being a single step process for collection, extraction, and stabilization of biological specimens at room temperature for long periods of time[4]. In addition to the known properties of the iSWAB™-ID buffer, this research examines the potential of using the iSWAB™-ID buffer to perform a single tube differential extraction. Various conditions of the iSWAB™-Cells-400 non-lytic buffer were also analyzed for their potential of stabilizing and protecting DNA stored within whole and intact cells under ambient conditions for up to 4 weeks. The following parameters of the iSWAB™-Cells-400 non-lytic buffer were tested: 1) ability to extract DNA from the intact cells with various methods after cells have been stored in the buffer; 2) compatibility with downstream polymerase chain reaction (PCR)-based analyses; and 3) ability to stabilize and store intact cells and their DNA for extended periods of time.
Overall, both epithelial cells (e-cells) and sperm cells were lysed within minutes of being added to the iSWAB™-ID buffer, eliminating the use of this buffer from being used for differential extraction. However, it was determined that the addition of dithiothreitol (DTT) aided in the recovery of both e-cell and sperm DNA. The iSWAB™-Cells-400 non-lytic buffer did not protect DNA for periods longer than a week, as degradation of DNA extracts was seen in cells stored in the buffer at room temperature for a week or more. Additional modifications to the iSWAB™-Cells-400 buffer should be explored before cells stored in this buffer can be successfully analyzed using short tandem repeat (STR) analysis.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/31294 |
| Date | 25 July 2018 |
| Creators | Yamamoto, Lisa |
| Contributors | Cotton, Robin W. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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