0
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 ...
Cotton Swabs
—  Widely used in crime
labs
—  Free of amplifiable
human DNA
—  Mattress design - Cotton
is wrapped aroun...
4N6FLOQTM Swabs
—  Made by Copan – patented
technology of nylon fibers
sprayed onto a plastic stick
—  Capillary action ...
NAOTM (Nucleic Acid Optimizer) Basket
—  Semi-permeable spin
basket developed by Copan
—  ‘valve’ opens only upon
centri...
Lymphocytes
Blood
Bacteria
http://vr-zone.com/articles/bacteria-communicate-to-
resist-antibiotics/43840.html
http://hemos...
Compare swab type
http://www.dreamstime.com/stock-photography-forensic-swab-image13609632
Vs
http://copanitalia.com/index-...
—  Compare 2 extractions
kits
¡  PrepFiler® Forensic DNA
Extraction Kit (Life Technologies,
Carlsbad, CA)
¡  DNA IQTM S...
—  With & w/o NAO
¡  Half of the samples
extracted with NAOs,
other half without
÷  In samples without
NAOs swab head
s...
Materials
—  DNA Samples
¡  Lymphocytes were isolated from fresh blood
sample using Histopaque® 1077 (Sigma-Aldrich, St....
Materials
—  Sample Preparation
¡  Cell count determined with
hemocytometer
¡  Dilution: ~80 cells/µL
¡  25µL aliquots...
Methods
—  Samples collected using
wet/dry method using
20µL 0.01% SDS
¡  All samples prepared,
collected and extracted
...
Issue: Flocked Swab with DNA IQ lysis buffer
—  When nylon flocked swabs were incubated in DNA IQTM
lysis buffer, extract...
Methods
—  Developed a modified protocol for DNA IQTM for
both swab types
¡  Original: Place swab and lysis buffer in tu...
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 ...
Prepfiler Vs. DNA IQ
•  Prepfiler: 14.21 ng
•  DNA IQ: 2.74 ng
•  419% increase with
Prepfiler
DNA IQ vs Prepfiler (w/NAO)...
Prepfiler Vs. DNA IQ
Number of Samples with Elevated Ct
DNA IQ Prepfiler
Cotton
Nylon
Flocked Cotton
Nylon
Flocked
Glass 0...
With NAO Vs. w/o NAO
Cotton/DNA IQ
Cotton/
Prepfiler
Nylon Flocked/
DNA IQ
Nylon Flocked/
Prepfiler
Glass NAO NAO NAO NAO
...
DNA IQ: Nylon
Flocked vs. Cotton
—  50% increase with Nylon
Flocked swab when
extracted with DNA IQ
—  980% increase wit...
Blood
Compare swab type
http://www.dreamstime.com/stock-photography-forensic-swab-image13609632
Vs
http://copanitalia.com/index-...
Materials
—  Blood is collected and stored at
-20˚C with EDTA
—  Conditions tested, 6 replicates
each
¡  5 µL of blood ...
Methods
—  Blood collected from glass the next day
¡  Wet/Dry technique (H2O not SDS 0.01%)
¡  All swabs allowed to air...
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
AverageDNAConcentration
(ng/µL)
Sample...
Results
0
0.1
0.2
0.3
0.4
0.5
0.6
20 minute lysis 40 minute lysis
AverageDNAConcentration
(ng/µL)
5 uL of Blood in Tube Ex...
Bacteria
Compare effect of bacterial contamination
Vs
Dried cotton swab Not dried flocked swab
specifically treated with
antibacter...
Materials
—  DNA Samples
¡  Cells were collected from 10 buccal
swabs
¡  Buccal cells were isolated in 10 mL
of 1x PBS
...
Materials
—  Sample Preparation
¡  Samples spotted on Plastic and allowed to
dry
÷  20 µL of buccal solution
¢  Collec...
Methods
—  Sample Prep repeated for 4 Conditions
¡  Incubation at 37˚C for 1 week
¡  Incubation at room temperature for...
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
Collecti...
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 ...
PODINI@GWU.EDU
AMD284@GWU.EDU
Questions?
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Cotton Swabs vs. 4N6FLOQSwabs™: A Comparative Study for Optimal DNA Recovery FROM Crime Scene Samples

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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)

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Transcript of "Cotton Swabs vs. 4N6FLOQSwabs™: A Comparative Study for Optimal DNA Recovery FROM Crime Scene Samples"

  1. 1. 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
  2. 2. 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).
  3. 3. 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
  4. 4. 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/
  5. 5. Lymphocytes Blood Bacteria http://vr-zone.com/articles/bacteria-communicate-to- resist-antibiotics/43840.html http://hemospat.com/terminology/index.php? cat=projected&sub=projectedhttp://biology.about.com/od/cellbiology/ss/lymphocytes.htm
  6. 6. Compare swab type http://www.dreamstime.com/stock-photography-forensic-swab-image13609632 Vs http://copanitalia.com/index-4.htm
  7. 7. —  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
  8. 8. —  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
  9. 9. 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
  10. 10. 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
  11. 11. 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
  12. 12. 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
  13. 13. 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
  14. 14. Human DNA Quantification Quantifiler® Human DNA Quantification Kit ABI 7000 System
  15. 15. Results
  16. 16. 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
  17. 17. 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
  18. 18. 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
  19. 19. 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
  20. 20. 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
  21. 21. Blood
  22. 22. Compare swab type http://www.dreamstime.com/stock-photography-forensic-swab-image13609632 Vs http://copanitalia.com/index-4.htm
  23. 23. 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/
  24. 24. 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
  25. 25. 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 AverageDNAConcentration (ng/µL) Sample DNA Recovery from Blood Samples Blood Directly in tube Nylon Cotton Results ?
  26. 26. Results 0 0.1 0.2 0.3 0.4 0.5 0.6 20 minute lysis 40 minute lysis AverageDNAConcentration (ng/µL) 5 uL of Blood in Tube Extraction 1.5 mL tube 2 mL tube
  27. 27. Bacteria
  28. 28. Compare effect of bacterial contamination Vs Dried cotton swab Not dried flocked swab specifically treated with antibacterial reagents
  29. 29. 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
  30. 30. 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
  31. 31. 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
  32. 32. 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 AverageDNAConcentration(ng/µL) 1 Week Incubation with Bacteria Nylon Cotton Directly in Tube
  33. 33. Results- to be determined —  Incubation at 37˚C for 2 weeks —  Incubation at room temperature for 2 weeks
  34. 34. Next Steps —  Replace lymphocytes with saliva —  Test new format: wider container/shorter swab
  35. 35. 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 A N D R E A C A R B O N A R O – L I F E T E C H N O L O G I E S Acknowledgements
  36. 36. PODINI@GWU.EDU AMD284@GWU.EDU Questions?
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