a comparative study of 4n6floqswabs™, cotton swabs, and swabbing solution

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A Comparative Study of 4N6FLOQSwabs™, Cotton Swabs, and Swabbing Solutions Dagmar Sweeney, Rodney Anderson Forensic Science Program Department of Biopharmaceutical Sciences University of Illinois at Chicago

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  1. 1. A Comparative Study of 4N6FLOQSwabs, Cotton Swabs, and Swabbing Solutions Dagmar Sweeney, Rodney Anderson Forensic Science Program Department of Biopharmaceutical Sciences University of Illinois at Chicago
  2. 2. Independent Study Continuation Cotton Swabs vs. 4N6FLOQSwabs: A Comparative Study for Optimal DNA Recovery FROM Crime Scene Samples given by Dr. Daniele Podini and Anna Dadhania, Department of Forensic Sciences, The George Washington University Compared: 2 swab types 2 extraction kits: PrepFiler Forensic DNA Extraction Kit (Life Technologies, Carlsbad, CA) and DNA IQ System (Promega, Madison, WI) NAO (Nucleic Acid Optimizer) Basket
  3. 3. Independent Study Continuation Cotton Swabs vs. 4N6FLOQSwabs: A Comparative Study for Optimal DNA Recovery FROM Crime Scene Samples given by Dr. Daniele Podini and Anna Dadhania, Department of Forensic Sciences, The George Washington University Results: 980% increase with 4N6FLOQSwabs swab when extracted with PrepFiler Forensic DNA Extraction Kit (Life Technologies, Carlsbad, CA)
  4. 4. Presentation Outline Introduction 1. DNA Recovery Comparison 2. The Influence of Swabbing Solution on DNA Recovery from a Nonporous Surface 3. Double Swab Technique 4. Sodium Dodecyl Sulfate Volume Optimization 5. Sample Collection from a Porous Surface 6. Effects of Time and Storing Conditions on DNA Recovery Discussion
  5. 5. Workflow for Casework Samples Collect Extract Quantify Amplify Detect & Analyze
  6. 6. Workflow for Casework Samples Collect Extract Quantify Amplify Detect & Analyze High Quantity DNA High Quality DNA
  7. 7. A Comparative Study of 4N6FLOQSwabs, Cotton Swabs, and Swabbing Solutions COPAN 4N6FLOQSWABS COTTON SWABSVs. 4N6FLOQSwabs and Cotton Swab, Dagmar Sweeney, McCrone Research Institute
  8. 8. Cotton Swabs Cotton fiber wrapped around wooden applicator 2 km of microfiber Inhibition of material release Hydrophilic Absorbs 25x its weight in water Sterile Widely used Cotton Swab, Dagmar Sweeney, McCrone Research Institute
  9. 9. Copan 4N6FLOQSwabs Sprayed-on nylon fibers on a solid molded plastic applicator 6 m microfiber Sample stays close to the surface Hydrophilic Capillary Action ETO-treated (Ethylene Oxide) Human DNA, DNase and RNase-free 4N6FLOQSwabs, Dagmar Sweeney, McCrone Research Institute
  10. 10. 1. DNA Recovery Comparison Questions asked: How high is DNA recovery from Nylon swabs? By how much is the DNA yield better or worse in comparison to cotton swabs? 4N6FLOQSwabs, Dagmar Sweeney, McCrone Research Institute
  11. 11. 1. DNA Recovery Comparison: Materials Fresh saliva collected and mixed with Tris EDTA buffer (TE buffer) Ratios of saliva to TE buffer tested 1:10 1:100 1:200 Applicators Copan Nylon Swabs Pur-Wraps Sterile Cotton Tipped Applicators
  12. 12. 1. DNA Recovery Comparison: Methods 75 L of each saliva solution applied directly on Nylon Swab (N=4) 75 L of each saliva solution applied directly on Cotton Swab (N=4) 75 L of TE buffer applied directly on Nylon Swab (N=1) 75 L of TE buffer applied directly on Cotton Swab (N=1)
  13. 13. 1. DNA Recovery Comparison: Methods All swabs allowed to air dry and stored at room temperature (RT) Swabs extracted after 1 week PrepFiler Forensic DNA Extraction Kit (Applied Biosystems) Automate Express DNA Extraction System (Applied Biosystems) Quantitation Quantifiler Trio DNA Quantification Kit (Applied Biosystems) 7500 Real Time PCR System (Applied Biosystems)
  14. 14. 1. DNA Recovery Comparison: Results 0 5 10 15 20 25 30 35 40 45 50 55 1:10 1:100 1:200 TotalDNAYield(ng) Saliva to TE Ratio Nylon Swabs Cotton Swabs * * 2 x more DNA recovered from nylon swabs Data presented as mean SE, N = 4. An unpaired, 2-tailed t-test was performed to compare average total DNA recovery from 4N6FLOQSwabsTM and cotton swabs. As indicated, * represents a P value of less than 0.01. *
  15. 15. 1. DNA Recovery Comparison: Hypothesis Nylon swabs: Sample stays close to the surface More surface area readily available for sample adhesion and release More sample released for analysis Nylon fiber from 4N6FLOQSwabs (20x magnification) Dagmar Sweeney, McCrone Research Institute
  16. 16. 2. The Influence of Swabbing Solution on DNA Recovery from Nonporous Surface Questions asked: How well nylon swabs perform when they are used to collect samples from a surface? Is there any interference with different swabbing solutions?4N6FLOQSwabs with water, Dagmar Sweeney, McCrone Research Institute
  17. 17. 2. The Influence of Swabbing Solution on DNA Recovery from Nonporous Surface The Influence of Swabbing Solutions on DNA Recovery from Touch Samples Sarah M. Thomasma, M.S. and David R. Foran, Ph.D. 2% sodium dodecyl sulfate (SDS) increased DNA yield
  18. 18. 2. The Influence of Swabbing Solution on DNA Recovery from Nonporous Surface: Materials Ceramic tile soaked in 10% bleach for 30 min, rinsed with water, autoclaved, air dried, and UV irradiated for 20 min on each side Fresh saliva collected and mixed with TE buffer in 1:200 ratio Swabbing Solutions 2% Sodium Dodecyl Sulfate (SDS) Deionized water (dH2O) Applicators Copan Nylon Swabs Pur-Wraps Sterile Cotton Tipped Applicators
  19. 19. 2. The Influence of Swabbing Solution on DNA Recovery from Nonporous Surface: Methods 50 L of the saliva/TE solution applied on the marked ceramic tile 50 L of the saliva/TE solution applied directly on nylon swabs and cotton swabs (N=3) Saliva deposits allowed to dry overnight Swabs moistened with 50 L of swabbing solution and passed 50x over dried saliva deposits (N=3)
  20. 20. 2. The Influence of Swabbing Solution on DNA Recovery from Nonporous Surface: Methods Swabs extracted immediately PrepFiler Forensic DNA Extraction Kit (Applied Biosystems) Automate Express DNA Extraction System (Applied Biosystems) Quantitation Quantifiler Trio DNA Quantification Kit (Applied Biosystems) 7500 Real Time PCR System (Applied Biosystems)
  21. 21. 2. The Influence of Swabbing Solution on DNA Recovery from Nonporous Surface: Results 0 0.5 1 1.5 2 2.5 Control Water 2% SDS TotalDNAYield(ng) Swabbing Solutions Nylon Swab Cotton Swab No significant difference in DNA yield with different swabbing solutions * Data presented as mean SE, N = 3. * denotes significantly higher DNA yields from 4N6FLOQSwabsTM than from cotton swabs and different swabbing solutions, based on ANOVA test. P value of less than 0.01.
  22. 22. 2. The Influence of Swabbing Solution on DNA Recovery from Nonporous Surface: Hypothesis SDS solution wet the swabbed surface more readily and thoroughly than did water alone Detergent seems to help to increase collected sample size but: Cotton swabs sample stays in cotton fibers Nylon swabs adhesion/release to/from nylon fibers and capillary action of the fibers unaffected 4N6FLOQSwabs with water (left) and 2% SDS (right), Dagmar Sweeney, McCrone Research Institute
  23. 23. 3. Double Swab Technique No significant difference in DNA yield with different swabbing solution. Observed residual liquid mainly when SDS used. How much DNA left behind? Cotton Swab with 2% SDS as a wetting agent, Dagmar Sweeney, McCrone Research Institute
  24. 24. 3. Double Swab Technique: Materials Ceramic tile soaked in 10% bleach for 30 min, rinsed with water, autoclaved, air dried, and UV irradiated for 20 min on each side Saliva collected and mixed with TE buffer in 1:200 ratio Swabbing Solutions 2% Sodium Dodecyl Sulfate (SDS) Deionized water (dH2O) Applicators Copan Nylon Swabs Pur-Wraps Sterile Cotton Tipped Applicators
  25. 25. 3. Double Swab Technique: Methods 50 L of saliva/TE solution applied on the marked ceramic tile 50 L of the saliva/TE solution applied directly on nylon swabs and cotton swabs (N=3) Saliva deposits allowed to dry overnight Swabs moistened with 50 L of swabbing solution and passed 50x over dried saliva deposits (N=3) A dry swab applied to absorb residual liquid
  26. 26. 3. Double Swab Technique: Methods Swabs extracted immediately and separately PrepFiler Forensic DNA Extraction Kit (Applied Biosystems) Automate Express DNA Extraction System (Applied Biosystems) Quantitation Quantifiler Trio DNA Quantification Kit (Applied Biosystems) 7500 Real Time PCR System (Applied Biosystems)
  27. 27. 3. Double Swab Technique: Results 0 0.5 1 1.5 2 2.5 Control Water 1st Swab Water 2nd Swab SDS 1st Swab SDS 2nd Swab TotalDNAYield(ng) Nylon Swab Cotton Swab 0.130 ng of DNA collected with the 2nd nylon swab / SDS Data presented as mean SE, N = 3. * denotes significantly higher DNA yields from 4N6FLOQSwabsTM than from cotton swabs and different swabbing solutions, based on ANOVA test. P value of less than 0.01.
  28. 28. 3. Double Swab Technique: Hypothesis SDS (detergent) loosens more sample from the surface Nylon swabs design allows for maximum uptake and release 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 Water 2nd Swab SDS 2nd Swab TotalDNAYield(ng) Nylon Swab Cotton Swab Data presented as mean SE, N = 3. * denotes significantly higher yields than water, based on independent samples t-test. (a P value of less than 0.01).
  29. 29. 4. SDS Volume Optimization Lots of residual liquid left behind when 50 L SDS used. What is the ideal SDS volume that leaves no residual liquid but allows for high sample collection? Nylon swab with 2% SDS as a swabbing solution, Dagmar Sweeney, McCrone Research Institute
  30. 30. 4. SDS Volume Optimization: Materials Ceramic tile soaked in 10% bleach for 30 min, rinsed with water, autoclaved, air dried, and UV irradiated for 20 min on each side Fresh saliva collected and mixed with TE buffer in 1:200 ratio Swabbing Solution 2% Sodium Dodecyl Sulfate (SDS) Applicator Copan Nylon Swabs
  31. 31. 4. SDS Volume Optimization: Methods 50 L of saliva / TE solution applied on the marked ceramic tile 50 L of saliva/TE solution directly on the control swab (N=3) Saliva deposits allowed to dry overnight Swabs moistened with 5, 10, 25, and 50 L of 2% SDS and passed 50x over dried saliva deposits (N=3)
  32. 32. 4. SDS Volume Optimization: Methods Swabs extracted immediately PrepFiler Forensic DNA Extraction Kit (Applied Biosystems) Automate Express DNA Extraction System (Applied Biosystems) Quantitation Quantifiler Trio DNA Quantification Kit (Applied Biosystems) 7500 Real Time PCR System (Applied Biosystems)
  33. 33. 4. SDS Volume Optimization: Results 0 0.5 1 1.5 2 2.5 3 3.5 Control 5 10 25 50 TotalDNAYield(ng) 2% SDS Volume (L) Less SDS Volume = Higher DNA Yield Data presented as mean SE, N = 3. ANOVA test was performed to compare average total DNA recovery from 4N6FLOQSwabsTM with various SDS volumes. * denotes significantly lower yields than 5 L, based on independent samples t-test, P value of less than 0.01.
  34. 34. 4. SDS Volume Optimization: Hypothesis Less SDS volume used to moisten nylon swabs increased DNA yield 5 L and 10 L: Only tip of the swab was moistened - enough to wet a sample area dry fibers collected remaining liquid 25 L - 50 L: Nearly the whole swab was wet no dry fibers to collect remaining liquid 4N6FLOQSwabs release samples even with soft pressure Nylon fibers from 4N6FLOQSwabs stored at 24C with 2% SDS as a swabbing solution (40 x magnification), Dagmar Sweeney, McCrone Research Institute
  35. 35. 5. Sample Collection from a Porous Surface Questions: How does 4N6FLOQSwabs compare to cotton swabs on porous surface? Is there any significant difference between 2% SDS and water on porous surface? Cotton fibers from a cotton swab stored at 24C with 2% SDS as a wetting agent (40 x magnification), Dagmar Sweeney, McCrone Research Institute
  36. 36. 5. Sample Collection from a Porous Surface: Materials Thin cloth soaked in 10% bleach for 30 min, rinsed with water, autoclaved, air dried, and UV irradiated for 20 min on each side Fresh saliva collected and mixed with TE buffer in 1:200 ratio Swabbing Solutions 2% Sodium Dodecyl Sulfate (SDS) Deionized Water Applicators Copan Nylon Swabs Pur-Wraps Sterile Cotton Tipped Applicators
  37. 37. 5. Sample Collection from a Porous Surface: Methods Cloth was elevated from the surface 50 L of saliva / TE solution applied on the marked cloth 50 L of saliva/TE solution directly on the control swab (N=3) Saliva deposits allowed to dry overnight Swabs moistened with 25 L of swabbing solution and passed 50x over dried saliva deposits (N=3)
  38. 38. 5. Sample Collection from a Porous Surface: Methods Swabs extracted immediately PrepFiler Forensic DNA Extraction Kit (Applied Biosystems) Automate Express DNA Extraction System (Applied Biosystems) Quantitation Quantifiler Trio DNA Quantification Kit (Applied Biosystems) 7500 Real Time PCR System (Applied Biosystems)
  39. 39. 5. Sample Collection from a Porous Surface: Results 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Control SDS Water TotalDNAYield(ng) Nylon Swab Cotton Swab Significantly more DNA recovered from cotton swabs Data presented as mean SE, N = 3. ANOVA test was performed to compare average total DNA recovery from 4N6FLOQSwabsTM to cotton swabs with different swabbing solutions.
  40. 40. 5. Sample Collection from a Cloth: Hypothesis Nylon swabs released lots of liquid upon a touch with the cloth surface Competitive behavior between nylon fibers and cloth porous fibers 0 0.005 0.01 0.015 0.02 0.025 SDS Water TotalDNAYield(ng) Nylon Swab Cotton Swab Data presented as mean SE, N = 3. An unpaired, 2-tailed t-test was performed to compare average total DNA recovery from 4N6FLOQSwabsTM and cotton swabs. * denotes significantly higher yields than nylon swabs based on independent samples t-test (P value of less than 0.05).
  41. 41. 6. Effects of Time and Storing Conditions on DNA Recovery Questions asked: DNA degradation? Bacterial contamination? 4N6FLOQSwabs stored at -20C with 2% SDS as wetting agent, Dagmar Sweeney, McCrone Research Institute
  42. 42. 6. Effects of Time and Storing Conditions on DNA Recovery: Materials Saliva collected and mixed with TE buffer in 1:10 ratio Applicators Copan Nylon Swabs Pur-Wraps Sterile Cotton Tipped Applicators
  43. 43. 6. Effects of Time and Storing Conditions on DNA Recovery: Methods Swabs moistened with 75 L of saliva / TE solution All swabs allowed to air dry Swabs stored at room temperature (RT) and freezing temperature (FT) (N=3) RT - app. 20C - 23C FT - app. - 20C
  44. 44. 6. Effects of Time and Storing Conditions on DNA Recovery: Methods Swabs extracted when air dried and after 3 months PrepFiler Forensic DNA Extraction Kit (Applied Biosystems) Automate Express DNA Extraction System (Applied Biosystems) Quantitation Quantifiler Trio DNA Quantification Kit (Applied Biosystems) 7500 Real Time PCR System (Applied Biosystems)
  45. 45. 6. Effects of Time and Storing Conditions on DNA Recovery: Results 0 10 20 30 40 50 60 TotalDNAYield(ng) DNA lost after 3 months at RT but not FT Data presented as mean SE, N = 3. ANOVA test was performed to compare average total DNA recovery from 4N6FLOQSwabsTM to cotton swabs under different storing conditions. * denotes significantly lower yields than FT environment, based on ANOVA and independent samples t-test.
  46. 46. 6. Effects of Time and Storing Conditions on DNA Recovery: Hypothesis Nylon swabs: Sample embedded close to the surface leads to better DNA recovery but also faster DNA degradation More sample is lost through handling Nylon fibers from 4N6FLOQSwabs stored at 24C with 2% SDS as a swabbing solution (40 x magnification) Dagmar Sweeney, McCrone Research Institute
  47. 47. STR Profiles Globalfiler PCR Amplification Kit (Life Technologies) Full STR profiles generated using GeneMapper ID-X 1.4 All tested samples provided full STR profiles
  48. 48. STR Profiles
  49. 49. Project Continuation Continue to observe effects of time and storing conditions on DNA recovery Effects of time and storing conditions on DNA recovery when detergent is used Examine other surfaces (jeans, leather, brick, wood, etc.) Set up ideal conditions for sample collection (swabbing solutions, swabbing solution volume)
  50. 50. Conclusion Pros: 4N6FLOQSwabs easy to handle No razor or scissors needed Low volume of a wetting agent needed to moisten swabs Outperforms cotton swabs when low count number DNA samples are collected from a nonporous surface.
  51. 51. Conclusion Cons: Sample easily released by minor pressure Transfer or storage containers should be rinsed with lysis buffer before extraction Competitive behavior between nylon and porous fibers of a porous surface Nylon fibers from 4N6FLOQSwabs (40 x magnification) Dagmar Sweeney, McCrone Research Institute
  52. 52. Acknowledgments Forensic Science Program, University of Illinois at Chicago Thermo Fisher Scientific / Life Technologies McCrone Research Institute, Chicago
  53. 53. References Anna Dadhania, Daniele Podini. Cotton Swabs vs. 4N6FLOQSwabs: A Comparative Study for Optimal DNA Recovery FROM Crime Scene Samples. Life Technologies 2013. B.C.M. Pang *, B.K.K. Cheung. Double swab technique for collecting touched evidence. Legal Medicine 2007; 9: 181-4. C. Lenz *, L.R. Flodgaard, B. Eriksen, N. Morling. Retrieval of DNA and genetic profiles from swabs taken inside cars. International Congress Series 2006; 1288: 5957. Graham Williams*, Manohar Pandre, Waseeh Ahmed, Emma Beasley, Emma Omelia, Damian World and Holly Yu. Evaluation of Low Trace DNA Recovery Techniques from Ridged Surfaces. J Forensic Res 2013, 4:4. Sarah M. Thomasma,1 M.S. and David R. Foran,2 Ph.D. The Influence of Swabbing Solutions on DNA Recovery from Touch Samples. J Forensic Sci, 2013, 58, 10.1111/1556- 4029.12036. Robert OBrien, Debra Figarell. Swab Collection Study. National Institute of Justice 2012.
  54. 54. Questions? Dagmar Sweeney [email protected] Rodney Anderson [email protected]