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

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

PODINI@GWU.EDU AMD284@GWU.EDU

Questions?