Maximizing the Amount of DNA Recovered: A Study of Mawi DNA Technologies iSWABTM-ID Collection Device for Forensic Science Application Boston University School of Medicine Program in Biomedical Forensic Sciences 72 E. Concord Street, Boston, MA 02118 Presentation By: Michelle K. Gordon, M.S. Problem Statement Recovery of DNA is extremely important Ability to maximize collection of DNA is limited by various issues
Structural design of swabs Field conditions and dry times Extraction procedures (>50% loss, Adamowicz et al. 2014) Ability to stabilize and protect the collected DNA at the point of collection, transit and storage  Adamowicz, M. S. et al (2014). PLoS ONE 9(12), 118. Mawi DNA Technologies iSWABTM-ID Collection Device Collection device design facilitates release of cells captured from any type of swab Prong System Proprietary lysis and DNA stabilizing buffer
Used at room temperature  US Patent No. 9,138,205 Goal/Objectives of the Research The purpose of the project was to define conditions and limitations of the use of the new collection device and associated (iSWAB TM) buffer system for forensic samples and subsequent DNA testing Experiments were designed to assess the ability to collect dried body fluid stains
to recover and lyse cells from different types and conditions of swabs to stabilize DNA to perform in downstream PCR amplification to produce quality STR profiles Methods Body fluids were donated anonymously A Hemocytometer was used to estimate concentrations of cellular suspensions Quantifiler Duo Quantification Kit on the 7500 Real-Time PCR system AmpFlSTR Identifiler Plus Kit and the Globalfiler Kit using the GeneAmp PCR System 9700 3130 Genetic Analyzer
GeneMapper ID-X JMP Pro v. 13 or Microsoft Excel for Statistics Optimized Dilution for Direct PCR iSWABTM buffer Concentration Result 0X Not Inhibited 0.1X
Not Inhibited 0.2X Not Inhibited 0.25X Partially Inhibited 0.3X Inhibited
Protocol Based On Buffer Concentration Optimization All experimental samples diluted to a 0.1X concentration of iSWABTM buffer prior to PCR amplification data confidence ease of calculations Test DNA Recovery: Wet and Dry Swabs Result: No Significant Difference in DNA Recovery Overall Average of Overall DNA Concentration Standard Deviation
Control 1.77 +/- 0.04 50L of cells directly in iSWABTM buffer 1.78 +/- 0.05 Test Efficiency of the Prong Mechanism
Prong Mechanism No Prong Mechanism Initial Eluted Initial Swab after elution
Eluted Swab after elution Results: Prong Mechanism Significantly Increase DNA Recovery Average % of Total ng of DNA 100% 90% 1.0
20.4 80% 12.0 31.5 70% 60% 50% 40% 78.6 30%
56.5 20% 10% 0% Prong Initial Swab after Elution No Prong Eluted Test Recovery from Dried Body Fluid Stains
Body Fluids Blood Combinations Cotton Swab moistened with iSWABTM buffer Nylon Flocked Swab moistened with iSWABTM buffer
Cotton Swab moistened with dH2O Nylon Flocked Swab moistened with dH2O Semen Saliva Results: Recovery from Dried Body Fluid Stains Comparison of DNA Recovery Using iSWABTM Buffer and
Silica Based Extraction Sample iSWABTM Buffer Extract Total DNA (ng) Average Total DNA (ng) 621 501
552 62 534 129 Silica Based Extract 150 207 162 40 Stability of DNA in iSWABTM Buffer Over 3-Month Period Average Total ng of DNA
Results: Stability of DNA in iSWABTM Buffer Over 3-Month Period Month 1 Month 2 Month 3 No DNA iSWABTM buffer Room Temperature TE buffer Frozen TE buffer
STR Profiles: 3 Months in iSWABTM Buffer at Room Temperature GlobalFiler Identifiler Plus Conclusions Use of the iSWABTM buffer enhances dried stain collection Swabs can be processed immediately or after being dried Mechanics of the device significantly enhance the recovery of cells from the swab Can lyse sperm, WBC and saliva cells Recovers >2 times more DNA than Silica Based Extractions Must dilute to a concentration of 0.2X or lower prior to PCR (experimental
data used 0.1X) PCR Amplification of 0.1X iSWABTM buffer produces full, high quality profiles Demonstrated DNA stability for up to 3 months at room temperature Acknowledgements Dr. Robin Cotton, Thesis Advisor Dr. Bassam El-Fahmawi from Mawi DNA Technologies Biomedical Forensic Science Program QUESTIONS? [email protected]
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