Automate Express to Streamline Differential Extraction process Presentation by Verdugo Regional Crime Laboratory (Glendale, CA) BodeWest

1. Differential extraction procedure utilizing
the AutoMate Express™ System
Rebecca Berlin, MS
Verdugo Regional Crime Laboratory
Glendale, CA

2. Glendale, CA 2010 census:
~190KLos Angeles County:

3. Glendale crime stats
Crime 2012 2013
Homicide 0 1
Rape 6 8
Robbery 108 75
Aggravated Assault 119 98
Property Crimes 3050 3201
In 2013, sexual assaults were only 0.2% of total UCR Part I crimes
and 4% of violent crimes in Glendale, CA .
Source: GPD Crime Statistics & Activity, 12/2013

4. That being said…
I have to do a differential extraction?!?!?!

5. Differential Organic Extraction
Figure 2.4 – modified from Butler, 2012.
+ Digest Buffer & pro K
Incubate 56°C – 1 hr
or 37°C – 2 hrs to O/N
Epithelial cell lysis
PCI
PCI
+ Digest Buffer, pro K & DTT
Incubate 56°C – 2 hrs
or 37°C – O/N
washes
Sperm cell lysis

6. 12 1
2
3
4
567
8
9
10
11
How much time are we spending?
Long incubation periods
Labor intensive process

7. Differential Organic Extraction
Figure 2.4 – modified from Butler, 2012.
+ Digest Buffer & pro K
Incubate 56°C – 1 hr
or 37°C – 2 hrs to O/N
Epithelial cell lysis PCI
PCI
+ Digest Buffer, pro K & DTT
Incubate 56°C – 2 hrs
or 37°C – O/N
washes
Sperm cell lysis

8. Automate Express™ Extraction System
• Two available forensic DNA extraction
kits
• PrepFiler Express™ Forensic DNA kit:
– Optimized for common forensic sample
types
– VRCL validated for touch, body fluids
(including semen), and hair roots
• PrepFiler Express BTA™ Forensic DNA
kit
– Optimized for bone, teeth, and adhesive-
containing samples (cigarettes, gum,
etc.)
– VRCL validated for adhesive-containing
samples

9. PrepFiler Express™ Forensic DNA Extraction Kit
• Components include:
– PrepFiler™ Lysis Buffer
– PrepFiler Express™ Cartridges
– PrepFiler Lysep™ columns and tubes
– Tips and Tip holders
• Protocol includes addition of 1.0M DTT to PrepFiler™ Lysis
Buffer (PLB)
• PLB + DTT added to each sample tube and incubated for 40
min @ 70°C, 750 rpm AVG CT ST DEV
Organic
Extraction
Case-Type Samples 28.19 0.14
Reagent Blanks 28.22 0.08
Robotic
extraction
Case-Type Samples 28.25 0.16
Reagent Blanks 28.28 0.08
Both procedures effective at removing inhibitors.

10. Differential Robotic Extraction
Figure 2.4 – modified from Butler, 2012.
+ Digest Buffer & pro K
Incubate 56°C – 1 hr
or 37°C – 2 hrs to O/N
Epithelial cell lysis
+ Digest Buffer, pro K & DTT
Incubate 56°C – 2 hrs
or 37°C – O/N
washes
Sperm cell lysis
??????
??????

11. Extraction Conditions
Epithelial Cell Lysis
- Blood and buccal samples incubated for 40 min @ 70°C, 750
rpm per previous validation and placed on AutoMate
Express™ Robot
- No significant difference detected for blood samples
- For buccal samples, digest buffer more effective extraction
buffer than PrepFiler Lysis Buffer
Sample Type
Digest Buffer (ng/µl) PrepFiler Lysis Buffer (ng/µl)
-DTT +DTT -DTT +DTT
Blood 1.382 1.945 1.208 1.759
Buccal 23.70 32.24 4.35 13.95

12. Extraction Conditions (cont.)
Sperm Cell Lysis
- Semen samples from two
donors were serially diluted
from neat to 1/8192 and dried
on cotton swabs
- ~1/2 of swab extracted
- More alleles and heterozygous
pairs observed with PLB
extraction
- Slightly higher average peak
heights and peak height ratios
observed with PLB extraction
Semen
Dilution
Digest Buffer w/ proK & DTT
Exp. No.
Obs.
No. Obs.
Avg. PH
(RFU)
No. Exp.
Het. Pairs
No. Obs.
Het. Pairs
Avg. PHR
Neat 96 96 1179 38 38 84.2
1/4 96 96 1542 38 38 83.0
1/16 96 96 1470 38 38 82.9
1/64 96 96 1246 38 38 83.4
1/256 96 85 2590 38 30 87.5
1/1024 96 84 439 38 30 76.6
1/4096 96 23 224 38 5 64.9
1/8192 96 21 135 38 1
Semen
Dilution
PrepFiler Lysis Buffer w/ DTT
Exp. No.
Obs.
No. Obs.
Avg. PH
(RFU)
No. Exp.
Het. Pairs
No. Obs.
Het. Pairs
Avg. PHR
Neat 96 96 1235 38 38 85.5
1/4 96 96 1265 38 38 85.3
1/16 96 96 1452 38 38 87.3
1/64 96 96 2176 38 38 85.4
1/256 64 64 3135 25 25 89.5
1/1024 96 86 814 38 31 83.8
1/4096 96 32 285 38 12 73.9
1/8192 96 30 244 38 10 71.8

13. Differential Robotic Extraction
Figure 2.4 – modified from Butler, 2012.
+ Digest Buffer & pro K
Incubate 70°C – 40 min,
750 rpm
Epithelial cell lysis
+ PrepFiler Lysis Buffer & DTT
Incubate 70°C – 40 min,
750 rpm
washes
Sperm cell lysis

14. Robotic vs Organic Extraction
Semen:blood mixture dilutions spotted on cotton; ~ 3x3 mm2 cutting
Organic procedure demonstrated
slightly higher quantitation values.
As expected, no detectable DNA in SF
fraction of neat blood sample.

15. Robotic vs Organic Extraction
No sperm fraction profile exhibited extraneous peaks attributable to the blood donor.
SF - 1:20 semen:blood mixture
Organic extraction Robotic extraction

16. Robotic vs Organic Extraction
Mixture
Ratio
Exp. No.
Obs.
Alleles
No. Obs. Alleles Avg. PH (RFU)
No. Exp.
Het Pairs
No. Obs. Het. Pairs Avg. PHR
Organic Robotic Organic Robotic Organic Robotic Organic Robotic
Neat Semen 128 128 128 922 948 52 52 52 80.9 83.4
1:1 128 128 128 923 957 52 52 52 82.7 86.4
1:3 128 128 128 846 1005 52 52 52 85.3 85.0
1:7 128 128 128 887 806 52 52 52 81.7 82.9
1:10 128 128 128 865 718 52 52 52 80.9 84.7
1:15 128 110† 128 875 611 52 41 52 80.9 80.4
1:20 128 128 126† 975 945 52 52 50 80.8 83.2
2:1 128 128 128 1028 1054 52 52 52 81.6 88.0
Differential extractions using the AutoMate Express™ system are comparable to
manual differential organic extractions.
† partial profile for one sample

17. Take Home Messages
• Digest buffer was a more effective lysis buffer than PrepFiler
Lysis Buffer for epithelial cell lysis.
• For mixed semen samples, the organic and robotic differential
extraction methods are effective in separating sperm and
non-sperm fractions.
– Differential robotic extraction reliably produced single donor male
profiles in the sperm fraction
• Use of the AutoMate Express™ System reduces the labor and
time invested in differential extractions
• Added benefit of reducing the potential of sample switching

18. Much obliged…
• Forensic Biology Unit
– Lisa Brewer, MS
– Becky Berlin, MS
– Catherine Nguyen, PhD
• VRCL Staff
• Glendale Police Department
Command Staff
Boss Lady
Boss Lady’s left handBoss Lady’s right
hand