Internal Developmental Validation of a DNA …...Internal Developmental Validation of a DNA Differential Extraction Protocol for Forensic Applications Allison N Miller, Meghan A Tory,

Internal Developmental Validation of a DNA Differential Extraction Protocol for Forensic

Applications

Allison N Miller, Meghan A Tory, Britton LF Morin Christian Westring, and Heather E McKiernan

Sexual Assaults

• Common Items in SA Kit

– Oral swabs

– Vaginal swabs

– Rectal swabs

• Typically mixture of epithelial and sperm cells

Differential Extraction

= sperm cell

= non-sperm cell

Differential Extraction

= sperm cell

= non-sperm cell

Issues with Differential Extractions

• Time-consuming

• Require several tube transfers

– Risk for contamination and/or sample loss

• Difficult to automate

• Use of harsh chemicals

– PCIA

Literature

Research Aims

• Design and validate an in-house differential extraction protocol

– Minimize carryover of sperm and epithelial DNA

– Minimize sperm loss

– Capable of being adapted to robotics

• Compare this protocol to four different extraction methods

• Assess various density liquids to minimize carryover:

– Diethyl Glutarate

– Dimethyl Glutarate

– 1-chloro-2-methyl-2-propanol*

Aim 1: Design in-house Protocol

Digest Buffer

Barrier Buffer

Sperm Pellet


Extraction Buffer +

Proteinase K Extraction

Buffer + DTT Lysis Buffer ATL Buffer

Diethyl Glutarate

NO SEPARATION

Diethyl Glutarate +

Butanol NO SEPARATION

Dimethyl Glutarate

NO SEPARATION

Dimethyl Glutarate +

Butanol NO SEPARATION


PCIA butanol

+

Barrier Buffers Tested

0

5

10

15

20

25

30

Modified PCIA DNA IQ

Ave

rage

% D

on

or

Alle

les

Det

ect

ed

fro

m M

ino

r C

on

trib

uto

r in

Sp

erm

Fra

ctio

n

Method Optimization

0

10

20

30

40

50

60

70

80

90

300µL Lysis Buffer+ 3µL DTT 300µL Lysis Buffer+ 20µL DTT

300µL Lysis Buffer+ 20µL DTT+10µL ProK

Tota

l DN

A R

eco

vere

d (

ng)

Average Total DNA Recovered from Spermatozoa Fraction


Sperm and Epithelial Cell Separation

Buffer Pro K Heat Barrier Buffer Wash

Sperm Fraction Buffer Heat

500µL extraction

buffer 5µL

56°C 40 min

100µL 500µL water

300µL lysis buffer + 3µL DTT

89°C 40 min

Sample Clean-Up

Filter Prep PCIA Butanol Filter Wash Spin RPM

Sperm Cell Elution Volume

Epithelial Cell Elution

Volume

100µL TE-4

300µL x2 200µL TE-4

5900

75µL 100µL

Aim 2: Comparison of Extraction Methods

1) Modified phenol:chloroform extraction + barrier buffer

2) Phenol:chloroform protocol

3) Promega Differex™ System

4) Promega DNA IQ + barrier buffer

5) Qiagen BioRobot EZ1 + barrier buffer

Comparison of Extraction Methods

Separation Phenol:

chloroform Promega Differex

System DNA IQ + Barrier

Buffer

EZ 1 BioRobot + Barrier buffer

Buffer 500μL Extraction Buffer+ 5μL ProK

400μL Digestion Buffer+ 4μL ProK

500μL Extraction Buffer+ 5μL ProK

500μL Extraction Buffer+ 5μL ProK

Incubation 56°C for 40 min 56°C for 1hr 56°C for 40 min 56°C for 40 min

Physical Separation

NONE 100μL Separation

Solution 100μL Diethyl

Glutarate 100μL Diethyl

Glutarate

Wash 500μL TNE 500μL Water 500μL Water 500μL Water

Sperm Fraction Buffer

350μL Extraction Buffer+ 20μL

DTT+ 10μL ProK

200μL Lysis Buffer+ 2μL DTT



Incubation 89°C for 40 min 70°C for 30 min 70°C for 30 min 89°C for 40 min

Comparison of Extraction Methods

Phenol: chloroform

Promega Differex System

Paramagnetic + Barrier Buffer

EZ 1 BioRobot + Barrier buffer

Clean Up

300μL PCIA (2x) 7μL resin 7μL resin

ROBOT

300μL Butanol 100μL Lysis Buffer+

1μL DTT 100μL Lysis Buffer+

1μL DTT

200μL TE-4 1x wash buffer(3 x) 1x wash buffer (3x)

65°C for 5 min 65°C for 5 min

Elution

Epithelial Cell Fraction-100μL

100μL 100μL 100μL

Sperm Cell Fraction-75μL

Sample Preparation

Semen (μL)

Whole Blood (μL)

Total Volume (μL)

1:1 300 300 600

1:10 54.5 545.5 600

1:100 6 594 600

Methods

Reserve Wash

0

20

40

60

80

100

120

140

160

180

1:1 1:10 1:100

Tota

l DN

A R

eco

vere

d (

ng)

Dilution (Semen:Blood)

Average Total DNA Recovered from Epithelial Fraction

Promega Differex

DNA IQ + Barrier Buffer

PCIA

Modified PCIA+ Barrier Buffer

Qiagen EZ1 + Barrier Buffer

*

*

*

*

0

200

400

600

800

1000

1200

1400

1600

1800

2000

1:1 1:10 1:100

Tota

l DN

A R

eco

vere

d (

ng)


Average Total DNA Recovered from Spermatozoa Fraction

Promega Differex

DNA IQ + Barrier Buffer

PCIA

Modified PCIA + Barrier Buffer Qiagen EZ1 + Barrier Buffer

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

0

10

20

30

40

50

60

70

80

90

1:1 1:10 1:100

Ave

rage

Pe

rce

nta

ge o

f M

ino

r A

llele

s D

ete

cte

d


Minor Alleles Detected in Epithelial Fraction

PCIA

Promega Differex

Modified PCIA + Barrier Buffer DNA IQ + Barrier Buffer


Promega Differex

Promega DNA IQ + Barrier

Buffer

Phenol: Chloroform Extraction

Modified Phenol:

Chloroform + Barrier Buffer


1:1 No Carryover

Detected No Carryover




Detected

1:10 No Carryover

Detected

1:100 No Carryover

Detected

Carryover Detected in Sperm Fraction

Sperm Hy-Liter™ Express

Fix Solution: 5 mins

Sample Preparation: 10 mins

Block: 15 mins

Sperm Head Stain: 15 mins

Mount

Reserved Wash

Issues Encountered with Sperm Hy-Liter™ Express

Positive Control 1:10 EZ 1 + Barrier Buffer

0

100

200

300

400

500

600

700

800

1:1 1:10 1:100

Ave

rage

# o

f Sp

erm


Average Number of Sperm Lost During Wash Steps

PCIA

PCIA + Barrier Buffer

Promega Differex

DNA IQ + Barrier Buffer Qiagen EZ1 + Barrier Buffer

Conclusions

Promega Differex

Promega DNA IQ +

Barrier Buffer

Phenol: Chloroform Extraction

Modified Phenol:

Chloroform + Barrier Buffer

Qiagen EZ1 + Barrier

Buffer

Maximize DNA Recovery

Minimize Carryover

Minimize Sperm Loss

Capable of being Adapted to Robotics

Acknowledgments

• Heather E. McKiernan, MSFS

• Meghan A. Troy, MSFS

• Britton Morin, MSFS

References 1) Hennekens, C. M., Cooper, E. S., Cotton, R. W., & Grgicak, C. M. (2013). The effects of differential extraction conditions on the

premature lysis of spermatozoa. Journal of Forensic Sciences, 58(3), 744–52. doi:10.1111/1556-4029.12098 2) Valgren C. and E. Edenberger, Evaluation of the DifferexTM System, Forensic Science International: Genetics Supplement

Series 1 (2008) 78-79. Valgren and Edenberger 3) Stray, J. J. Liu, M. Brevnov, and J. Shewale, Extraction of DNA from Forensic Biological Samples for Genotyping. CRC Press

(2013). 39-64. 4) Norris, J. V, Manning, K., Linke, S. J., Ferrance, J. P., & Landers, J. P. (2007). Expedited, chemically enhanced sperm cell

recovery from cotton swabs for rape kit analysis. Journal of Forensic Sciences, 52(4), 800–5. doi:10.1111/j.1556-4029.2007.00453.x

5) Stray, J. J. Liu, M. Brevnov, and J. Shewale, Extraction of DNA from Forensic Biological Samples for Genotyping. CRC Press (2013). 39-64.

6) Lounsbury, J., J. Bienvenue, J Landers, Sample-to-Result STR Genotyping System : Potential and Status (2012). Forensic Science Review 24(2):123–142.

7) Tackling the Backlog Using Automated Differential Extraction. (n.d.). Retrieved April 18, 2014, from http://www.forensicmag.com/articles/2008/04/tackling-backlog-using-automated-differential-extraction

8) Chen, J., Kobilinsky, L., Wolosin, D., Shaler, R., & Baum, H. (1998). A physical method for separating spermatozoa from epithelial cells in sexual assault evidence. Journal of Forensic Sciences, 43(1), 114–8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9456531

9) Giles, Robert C. (2008). Improved Methods for the Elution and Extraction of Spermatazoa from Sexual Assault Swabs. Forensic Magazine. Retrieved from http://www.forensicmag.com/articles/2008/04/improved-methods-elution-and-extraction-spermatozoa-sexual-assault-swabs

11) Garvin, A. M., Fischer, A., Schnee-Griese, J., Jelinski, A., Bottinelli, M., Soldati, G., … Madrid, M. (2012). Isolating DNA from sexual assault cases: a comparison of standard methods with a nuclease-based approach. Investigative Genetics, 3(1), 25. doi:10.1186/2041-2223-3-25

12) Colussi, A., Viegas, M., Beltramo, J., & Lojo, M. (2009). Efficiency of DNA IQ System® in recovering semen DNA from cotton swabs. Forensic Science International: Genetics Supplement Series, 2(1), 87–88. doi:10.1016/j.fsigss.2009.09.031

13) Tsukada, K., Asamura, H., Ota, M., Kobayashi, K., & Fukushima, H. (2006). Sperm DNA extraction from mixed stains using the DifferexTM System. International Congress Series, 1288, 700–703. doi:10.1016/j.ics.2005.12.059

http://www.ncbi.nlm.nih.gov/pubmed/9456531

http://www.forensicmag.com/articles/2008/04/improved-methods-elution-and-extraction-spermatozoa-sexual-assault-swabs

















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Internal Developmental Validation of a DNA …...Internal Developmental Validation of a DNA Differential Extraction Protocol for Forensic Applications Allison N Miller, Meghan A Tory,

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