cotton swabs vs. 4n6floqswabs™: a comparative study for optimal dna recovery from crime scene...
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Cotton Swabs vs. 4N6FLOQSwabs™: A Comparative Study for Optimal DNA Recovery FROM Crime Scene Samples given by Dr. Daniele Podini and Anna Dadhania (2013 Life Technologies Forensic Seminar Series)TRANSCRIPT
A N N A D A D H A N I A D A N I E L E P O D I N I
D E P A R T M E N T O F F O R E N S I C S C I E N C E S T H E G E O R G E W A S H I N G T O N U N I V E R S I T Y
M I C H E L L E N E L S O N G L O R I A N N A C A V E S
R E B E C C A S A N T I A G O
Cotton Swabs vs. 4N6FLOQSwabs™: A Comparative Study for Optimal DNA Recovery
FROM Crime Scene Samples
Cotton Swabs
� Widely used in crime labs
� Free of amplifiable human DNA
� Mattress design - Cotton
is wrapped around a wooden stick ¡ Possible inhibition of
material release Probst et al., Appl. Environ. Microbiol. 76(15):5148-5158 (2010).
4N6FLOQTM Swabs
� Made by Copan – patented technology of nylon fibers sprayed onto a plastic stick
� Capillary action for absorption of samples
� Nylon fibers allow for easy release of material
� EtO treated – inhibitor free, DNase, RNase, and amplifiable Human DNA
Impact Nurse website. Accessed 17 Feb 2013. http://www.impactednurse.com/?p=1195
NAOTM (Nucleic Acid Optimizer) Basket
� Semi-permeable spin basket developed by Copan
� ‘valve’ opens only upon centrifugation
� Incubation can take place within NAO
� Reduces steps that expose
samples to contamination Copan Flock Technologies website. Accessed 12 Feb 2013. http://www.copanflocktech.com/index.php/prod/sampleprep/nucleic/
Lymphocytes Blood
Bacteria
http://vr-zone.com/articles/bacteria-communicate-to-resist-antibiotics/43840.html
http://hemospat.com/terminology/index.php?cat=projected&sub=projected http://biology.about.com/od/cellbiology/ss/lymphocytes.htm
Compare swab type
http://www.dreamstime.com/stock-photography-forensic-swab-image13609632
Vs
http://copanitalia.com/index-4.htm
� Compare 2 extractions
kits ¡ PrepFiler® Forensic DNA
Extraction Kit (Life Technologies, Carlsbad, CA)
¡ DNA IQTM System (Promega, Madison, WI)
� Both kits use magnetic bead/particle technology to bind DNA
Vs
Compare extraction kit
� With & w/o NAO
¡ Half of the samples extracted with NAOs, other half without ÷ In samples without
NAOs swab head squeezed against side of tube after incubation.
Compare use of NAO
Materials
� DNA Samples ¡ Lymphocytes were isolated from fresh blood
sample using Histopaque® 1077 (Sigma-Aldrich, St. Louis, MO)
÷ Lymphocytes & mononuclear cells remain at Histopaque/plasma interface
¡ Chose lymphocytes because they could be easily counted ÷ Theoretically use cell count to calculate amount
of DNA being spotted ÷ Amount of DNA recovered/amount
spotted=percent recovery
Materials
� Sample Preparation ¡ Cell count determined with
hemocytometer ¡ Dilution: ~80 cells/µL ¡ 25µL aliquots of cell solution
spotted onto substrate and allowed to dry (~2000 cells = ~12ng DNA)
¡ All samples run in triplicate
� Substrates ¡ Glass, leather, unfinished
wood, plastic, metal and gun grips
Methods
� Samples collected using wet/dry method using 20µL 0.01% SDS ¡ All samples prepared,
collected and extracted same day
� Swab heads removed ¡ Cotton swabs required use
of razor ¡ Nylon flocked swab has
break point http://www.copaninnovation.com/index.php
Issue: Flocked Swab with DNA IQ lysis buffer
� When nylon flocked swabs were incubated in DNA IQTM lysis buffer, extraction was completely inhibited ¡ Occurred in samples processed with and without NAO
� Indicates an incompatibility between this kit and the nylon flocked swabs ¡ Source of incompatibility unknown due to proprietary nature of
products
� DNA IQTM affected by:
¡ Denim dyes ¡ Hemastix® reagents
http://www.dreamstime.com/royalty-free-stock-photos-pocket-detail-dirty-blue-jeans-image1531528
http://www.bvda.com/EN/sect6/en_6_1a.html
Methods
� Developed a modified protocol for DNA IQTM for both swab types
¡ Original: Place swab and lysis buffer in tube and incubate at 70°C for 30 min
¡ Modified: Place swab and lysis buffer in tube, vortex at max speed for 30 seconds, centrifuge, remove swab/NAO containing swab, incubate filtrate at 70°C for 30 min
Human DNA Quantification
Quantifiler® Human DNA Quantification Kit ABI 7000 System
Results
Nylon Flocked Vs. Cotton
� Nylon Flocked: 14.65 ng
� Cotton: 2.30 ng
� 538% increase with Nylon Flocked swabs
Nylon vs Cotton (w/NAO)
DNA IQ Prepfiler
Glass Nylon Flocked Nylon Flocked
Leather Nylon Flocked Nylon Flocked
Wood Nylon Flocked Nylon Flocked
Plastic Cotton Nylon Flocked
Knife Cotton Nylon Flocked
Gun Nylon Flocked Cotton
Prepfiler Vs. DNA IQ
• Prepfiler: 14.21 ng
• DNA IQ: 2.74 ng
• 419% increase with Prepfiler
DNA IQ vs Prepfiler (w/NAO)
Cotton Nylon
Flocked
Glass DNA IQ DNA IQ
Leather Prepfiler Prepfiler
Wood DNA IQ Prepfiler
Plastic Prepfiler Prepfiler
Knife Prepfiler Prepfiler
Gun Prepfiler DNA IQ
Prepfiler Vs. DNA IQ
Number of Samples with Elevated Ct
DNA IQ Prepfiler
Cotton Nylon
Flocked Cotton Nylon
Flocked
Glass 0 2 0 0 Leather 3 0 0 0 Wood 1 2 0 0 Plastic 0 2 0 0 Knife 0 0 0 0 Gun 1 1 0 0
With NAO Vs. w/o NAO
Cotton/DNA IQ Cotton/ Prepfiler
Nylon Flocked/DNA IQ
Nylon Flocked/Prepfiler
Glass NAO NAO NAO NAO
Leather NAO No NAO NAO NAO
Wood NAO No NAO NAO No NAO
Plastic NAO NAO NAO NAO
Knife No NAO No NAO No NAO NAO
Gun NAO NAO NAO No NAO
� Average amount of DNA recovered with NAO: 3.96 ng
� Without NAO: 2.67 ng
� 48% increase with the use of NAO
DNA IQ: Nylon Flocked vs. Cotton
� 50% increase with Nylon Flocked swab when extracted with DNA IQ
� 980% increase with Nylon Flocked swab when extracted with Prepfiler
Prepfiler: Nylon Flocked vs. Cotton
Blood
Compare swab type
http://www.dreamstime.com/stock-photography-forensic-swab-image13609632
Vs
http://copanitalia.com/index-4.htm
Materials
� Blood is collected and stored at -20˚C with EDTA
� Conditions tested, 6 replicates each ¡ 5 µL of blood directly in tube ¡ 5 µL of blood spotted on glass and
collected with Nylon swab ¡ 5 µL of blood spotted on glass and
collected with Cotton swab ¡ 5 µL of blood spotted directly on Nylon
Swab ¡ 5 µL of blood spotted directly on
Cotton swab
http://www.npsg.uwaterloo.ca/people/daniel/rendering/
Methods
� Blood collected from glass the next day ¡ Wet/Dry technique (H2O not SDS 0.01%) ¡ All swabs allowed to air dry and stored at RT
� Swabs extracted after 2 weeks � Blood directly in tube extracted
immediately ¡ PrepFiler® Forensic DNA Extraction Kit
� Quantitation ¡ Quantifiler® Human DNA Quantification Kit
¡ ABI PRISM® 7000 Sequence Detection System
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Blood on Glass Blood on Swab Blood Directly in tube
Ave
rage
DN
A C
once
ntra
tion
(
ng/µ
L)
Sample
DNA Recovery from Blood Samples
Blood Directly in tube
Nylon
Cotton
Results
?
Results
0
0.1
0.2
0.3
0.4
0.5
0.6
20 minute lysis 40 minute lysis
Ave
rage
DN
A C
once
ntra
tion
(n
g/µL
)
5 uL of Blood in Tube Extraction 1.5 mL tube
2 mL tube
Bacteria
Compare effect of bacterial contamination
Vs
Dried cotton swab Not dried flocked swab specifically treated with antibacterial reagents
Materials
� DNA Samples ¡ Cells were collected from 10 buccal
swabs ¡ Buccal cells were isolated in 10 mL
of 1x PBS � Bacterial cells
¡ Bacteria collection ÷ Fingers, Forensic Department door
knob, restroom door knob, and cheek swab
¡ Colonies grown on agar plates ¡ Picked colonies from each plate
grown in solution of LB broth
Materials
� Sample Preparation ¡ Samples spotted on Plastic and allowed to
dry ÷ 20 µL of buccal solution
¢ Collected using nylon and cotton swabs using wet/dry technique
¢ Wetted with bacterial suspension ¡ Samples spotted directly onto swabs
÷ 20 µL of buccal solution ÷ With or without bacteria ÷ Spotted on nylon and cotton swabs
¡ Samples spotted directly into 1.5 mL tubes ÷ 20 µL of buccal solution
Methods
� Sample Prep repeated for 4 Conditions ¡ Incubation at 37˚C for 1 week ¡ Incubation at room temperature for 1 week ¡ Incubation at 37˚C for 2 weeks ¡ Incubation at room temperature for 2 weeks
� Extraction ¡ PrepFiler® Forensic DNA Extraction Kit
� Quantitation ¡ Quantifiler® Human DNA Quantification Kit
¡ ABI PRISM® 7000 Sequence Detection System
Results
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Collection, Dried, RT
Collection, Dried, 37˚C
Collection, Not Dried, RT
Collection, Not Dried,
37˚C
Directly on Swab, Dried,
RT
Directly on Swab, Dried,
RT, No bacteria
Directly in tube, Dried,
RT, No bacteria
Ave
rage
DN
A C
once
ntra
tion
(ng
/µL)
1 Week Incubation with Bacteria
Nylon
Cotton
Directly in Tube
Results- to be determined
� Incubation at 37˚C for 2 weeks � Incubation at room temperature for 2 weeks
Next Steps
� Replace lymphocytes with saliva � Test new format: wider container/shorter swab
M I C H E L L E N E L S O N G L O R I A N N A C A V E S
R E B E C C A S A N T I A G O A N N A D A D H A N I A
S A N T I N A C A S T R I C I A N O – C O P A N I T A L I A
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Acknowledgements
[email protected] [email protected]
Questions?