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Internal Developmental Validation of a DNA …...Internal Developmental Validation of a DNA...
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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
Aim 1: Design in-house Protocol
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
Aim 1: Design in-house Protocol
PCIA butanol
+
Barrier Buffers Tested
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Modified PCIA DNA IQ
Ave
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% D
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Alle
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Det
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Sp
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Method Optimization
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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
Aim 1: Design in-house Protocol
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
300μL Lysis Buffer+ 3μL DTT
300μL Lysis Buffer+ 3μL 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
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1:1 1:10 1:100
Tota
l DN
A R
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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
*
*
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0
200
400
600
800
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1200
1400
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2000
1:1 1:10 1:100
Tota
l DN
A R
eco
vere
d (
ng)
Dilution (Semen:Blood)
Average Total DNA Recovered from Spermatozoa Fraction
Promega Differex
DNA IQ + Barrier Buffer
PCIA
Modified PCIA + Barrier Buffer Qiagen EZ1 + Barrier Buffer
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5.00
10.00
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30.00
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1:1 1:10 1:100
Ave
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Dilution (Semen:Blood)
Minor Alleles Detected in Epithelial Fraction
PCIA
Promega Differex
Modified PCIA + Barrier Buffer DNA IQ + Barrier Buffer
Qiagen EZ1 + Barrier Buffer
Promega Differex
Promega DNA IQ + Barrier
Buffer
Phenol: Chloroform Extraction
Modified Phenol:
Chloroform + Barrier Buffer
Qiagen EZ1 + Barrier Buffer
1:1 No Carryover
Detected No Carryover
Detected No Carryover
Detected 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
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1:1 1:10 1:100
Ave
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# o
f Sp
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Dilution (Semen:Blood)
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
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