A two-step protocol has been devised as a rapid and selective alternative to conventional differential extraction techniques with an increased recovery of DNA. The protocol involves pressure cycling with the Barocycler® NEP 2320 from Pressure Biosciences. Inc. in alkaline conditions for epithelial cell lysis and removal. This step is followed by alkaline lysis at 95º C for extraction of sperm cell DNA. At 1:1 or 2:1 female to male cell ratios, high selectivity and complete separation can be achieved. But at higher ratios, male allelic dropout is observed. This protocol has been modified to generate a clean male profile at a 20:1 cell ratio through optimization of NaOH concentration and inclusion of an additional pressure cycling step. Validation studies have been performed to assess the efficiency of this method under various conditions. An additional immunomagnetic cell capture pretreatment allowed for nearly complete separation at cell ratios of up to 200:1.
| Identifer | oai:union.ndltd.org:fiu.edu/oai:digitalcommons.fiu.edu:etd-3981 |
| Date | 04 November 2016 |
| Creators | Martinez, Vanessa |
| Publisher | FIU Digital Commons |
| Source Sets | Florida International University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | FIU Electronic Theses and Dissertations |
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