Application of Pressure Cycling Technology (PCT) in Differential Extraction Deepthi Nori, MFS; Dr. Bruce R. McCord, PhD Department of Chemistry International Forensic Research Institute Florida International University
Overview Pressure Cycling Technology (PCT) Differential Extraction Objective Experimental design Results Conclusions
Pressure Cycling Technology Barocycler NEP2320 (Pressure BioSciences Inc.) User controlled variables: a) Pressure (5-45k psi) b) Holding time at ambient pressure (T1) c) Holding time at target pressure (T2) d) No. of cycles (1-99) e) Temperature PULSE TM tubes (up to 300 µl) MicroTubes (50-150 µl) Pressure BioSciences Inc.
Differential Extraction Sexual assault casework Separation of female-derived sperm cells and epithelial cells Sperm + Epithelial cells SDS. EDTA, Proteinase K Incubate at 37ºC Lysed Epithelial cells- DNA Sperm cell pellet Sperm pellet SDS. EDTA, Proteinase K + DTT DTT lyses sperm heads Male DNA fraction Male DNA profile
Objective Conventional Differential Extraction Digestion of Epithelial cells Isolation of Sperm cells Pressure-based extraction Intact Epithelial cells Selective lysis of Sperm cells
PCT- Microscopic studies Microscopic visualization of PCT-treated cells stained with 0.4% Trypan blue solution Trypan blue staining - dye exclusion method Cells with a compromised membrane integrity are stained dark blue Vaginal epithelial cells Sperm cells
Experimental design SAMPLES Sperm cells and Epithelial cells suspended in 1X PBS buffer (ph 7.4) CELL LYSIS Pressure cycling using Barocycler NEP 2320 QUANTITATION Real-time PCR analysis using Plexor HY system (Promega Corporation) PURIFICATION Phenol-Chloroform- Isoamyl alcohol extraction
Dose Response curve Total DNA in sample- Standard Phenol/Chloroform and Proteinase K extraction Low recovery * Average value of triplicates Results presented at 21 st International Symposium on Human Identification
Maximize DNA recovery Increase in temperature 40º C 50º C 60º C Individual samples Addition of detergent 2% Sarkosyl 2% Sodium dodecyl sulfate (SDS) 2% Tween-20 45000 psi 60 cycles Addition of reducing agent Dithiothreitol (DTT)
% DNA Recovery Effect of PCT on treated cells 18 16 14 12 10 8 6 4 2 0 Sperm cells Vaginal Epithelial cells * Average value of triplicates 45000 psi, 60 cycles Results presented at 21 st International Symposium on Human Identification
% DNA Recovery Dithiothreitol (DTT) studies 90 80 70 60 50 40 30 20 10 0 20 mm DTT Sperm cells 40 mm DTT 60 mm DTT 80 mm DTT 45000 psi, 60 cycles Vaginal epithelial cells Selectivity needs improvement 100 mm DTT * Average value of triplicates
Tris(2-carboxyethyl)phosphine (TCEP) Soluble in water More effective than DTT Odorless Selectively reduces disulfide bonds over a broad ph range Stable- resistant to air oxidation TCEP is not particularly stable in phosphate buffers Chemical Formula: Molecular Weight: C 9 H 16 O 6 PCl 286.6 g/mol Thermo Fisher Scientific Inc.
% DNA Recovery TCEP studies 70 60 50 40 30 20 10 0 Increased recovery 10 mm TCEP Increase in selectivity 20 mm TCEP 30 mm TCEP 40 mm TCEP 50 mm TCEP 45000 psi, 60 cycles Sperm cells (Control- 6.23 ng/µl) Vaginal Epithelial cells (Control- 4.03 ng/µl) * Average value of triplicates
% DNA Recovery TCEP studies 18 16 14 12 10 8 6 4 2 0 13.3 0.55 0.35 25K psi, 60 45K psi, 60 cycles cycles 20 mm TCEP Sperm cells (Control- 32.7 ng/µl) Vaginal Epithelial cells (Control- 40.3 ng/µl) Selectivity reproducible at higher concentrations of 14 sample 17.14 * Average value of triplicates 8.4 0.3 0.37 25k psi, 60 45k psi, 60 cycles cycles 40 mm TCEP
STR profile PowerPlex 16 HS (Promega Corporation) Sperm DNA control Sperm DNA- 20 mm TCEP + PCT (25K psi)
Conclusions Pressure Cycling Technology (PCT) - Potential application in differential extractions Tris (2-carboxyethyl) phosphine (TCEP) High selectivity Improved recovery Selective detection of sperm cells Successful amplification of sperm DNA recovered from pressure lysis Future work- Mixtures and dried stains
Acknowledgements Pressure BioSciences Inc. Matthew Potter: Vice President of Global Sales Nate Lawrence, PhD: Vice President of Marketing Alex Lazarev, PhD: Vice President of R&D Vera Gross, PhD: Senior Scientist Promega Corporation Len Goren, Promega Genetic Identity Director Broward Sheriff s Office DNA Laboratory Dr. George Duncan