Differential extraction procedure utilizing the AutoMate Express™ System

Rebecca Berlin, MSVerdugo Regional Crime Laboratory

Glendale, CA

Glendale, CA2010 census: ~190KLos Angeles County:

Glendale crime statsCrime 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

That being said…

I have to do a differential extraction?!?!?!

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

12 12

3

4567

8

9

1011

How much time are we spending?

Long incubation periodsLabor intensive process

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

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

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.

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

??????

??????

Extraction ConditionsEpithelial 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 TypeDigest 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

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. PairsNo. Obs. Het. Pairs Avg. PHR

Neat 96 96 1179 38 38 84.21/4 96 96 1542 38 38 83.0

1/16 96 96 1470 38 38 82.91/64 96 96 1246 38 38 83.4

1/256 96 85 2590 38 30 87.51/1024 96 84 439 38 30 76.61/4096 96 23 224 38 5 64.91/8192 96 21 135 38 1

Semen Dilution

PrepFiler Lysis Buffer w/ DTT

Exp. No. Obs. No. Obs. Avg. PH

(RFU)No. Exp.

Het. PairsNo. Obs. Het. Pairs Avg. PHR

Neat 96 96 1235 38 38 85.51/4 96 96 1265 38 38 85.3

1/16 96 96 1452 38 38 87.31/64 96 96 2176 38 38 85.4

1/256 64 64 3135 25 25 89.51/1024 96 86 814 38 31 83.81/4096 96 32 285 38 12 73.91/8192 96 30 244 38 10 71.8

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

Robotic vs Organic ExtractionSemen: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.

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

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

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

Much obliged…

• Forensic Biology Unit– Lisa Brewer, MS– Becky Berlin, MS– Catherine Nguyen, PhD

• VRCL Staff• Glendale Police Department

Command Staff

Boss Lady

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