a comparative study of 4n6floqswabs™, cotton swabs, and swabbing solution
TRANSCRIPT
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
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
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)
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
Workflow for Casework Samples
Collect Extract Quantify Amplify Detect & Analyze
Workflow for Casework Samples
Collect Extract Quantify Amplify Detect & Analyze
High Quantity DNA High Quality DNA
A Comparative Study of 4N6FLOQSwabs™, Cotton Swabs, and Swabbing Solutions
COPAN 4N6FLOQSWABS™ COTTON SWABS Vs.
4N6FLOQSwabs™ and Cotton Swab, Dagmar Sweeney, McCrone Research Institute
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
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
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
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
1. DNA Recovery Comparison: Methods
75 µL of each saliva solution applied directly on Nylon Swab (N=4)
75 µL of each saliva solution applied directly on Cotton Swab (N=4)
75 µL of TE buffer applied directly on Nylon Swab (N=1)
75 µL of TE buffer applied directly on Cotton Swab (N=1)
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)
1. DNA Recovery Comparison: Results
05
10152025303540455055
1:10 1:100 1:200
Tota
l DN
A Yi
eld
(ng)
Saliva to TE Ratio
Nylon SwabsCotton 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.
*
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
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
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
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
2. The Influence of Swabbing Solution on DNA Recovery from Nonporous Surface: Methods
50 µL of the saliva/TE solution applied on the marked ceramic tile
50 µL 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 µL of swabbing solution and passed 50x
over dried saliva deposits (N=3)
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)
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
Tota
l DN
A Yi
eld
(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.
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
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
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
3. Double Swab Technique: Methods
50 µL of saliva/TE solution applied on the marked ceramic tile
50 µL 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 µL of swabbing solution and passed 50x
over dried saliva deposits (N=3)
A dry swab applied to absorb residual liquid
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)
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
Tota
l DN
A Yi
eld
(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.
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
Tota
l DN
A Yi
eld
(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).
4. SDS Volume Optimization
Lots of residual liquid left behind when 50 µL 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
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
4. SDS Volume Optimization: Methods
50 µL of saliva / TE solution applied on the marked ceramic tile
50 μL 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 µL of 2% SDS and
passed 50x over dried saliva deposits (N=3)
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)
4. SDS Volume Optimization: Results
0
0.5
1
1.5
2
2.5
3
3.5
Control 5 10 25 50
Tota
l DN
A Yi
eld
(ng)
2% SDS Volume (µL)
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 μL, based on independent samples t-test, P value of less than 0.01.
4. SDS Volume Optimization: Hypothesis Less SDS volume used to
moisten nylon swabs increased DNA yield
5 μL and 10 μL: Only tip of the swab was moistened - enough to wet a sample area – dry fibers collected remaining liquid
25 μL - 50 μL: 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 24˚C with 2% SDS as a swabbing solution (40 x magnification), Dagmar Sweeney, McCrone Research Institute
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 24˚C with 2% SDS as a wetting agent (40 x magnification), Dagmar Sweeney, McCrone Research Institute
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
5. Sample Collection from a Porous Surface: Methods
Cloth was elevated from the surface
50 µL of saliva / TE solution applied on the marked cloth
50 μL of saliva/TE solution directly on the control swab (N=3)
Saliva deposits allowed to dry overnight
Swabs moistened with 25 µL of swabbing solution and
passed 50x over dried saliva deposits (N=3)
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)
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
Tota
l DN
A Yi
eld
(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.
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
Tota
l DN
A Yi
eld
(ng)
Nylon SwabCotton 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).
6. Effects of Time and Storing Conditions on DNA Recovery
Questions asked:
DNA degradation?
Bacterial contamination?
4N6FLOQSwabs™ stored at -20˚C with 2% SDS as wetting agent, Dagmar Sweeney, McCrone Research Institute
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
6. Effects of Time and Storing Conditions on DNA Recovery: Methods
Swabs moistened with 75 µL of saliva / TE solution
All swabs allowed to air dry
Swabs stored at room temperature (RT) and
freezing temperature (FT) (N=3)
RT - app. 20˚C - 23˚C
FT - app. - 20˚C
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)
6. Effects of Time and Storing Conditions on DNA Recovery: Results
0
10
20
30
40
50
60
Tota
l DN
A Yi
eld
(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.
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 24˚C with 2% SDS as a swabbing solution (40 x magnification) Dagmar Sweeney, McCrone Research Institute
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
STR Profiles
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)
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.
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
Acknowledgments
Forensic Science Program, University of Illinois at Chicago
Thermo Fisher Scientific / Life Technologies
McCrone Research Institute, Chicago
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: 595–7.
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 O’Brien, Debra Figarell. Swab Collection Study. National Institute of Justice 2012.