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Forensic Sample Processing using a Robotic Workstation: Automated Paper-Based Spotting of Whole
Blood Convicted Offender Samples and High Throughput DNA Isolation for STR Analysis
Michael Biondi1, Christopher Palaski1, Christine Tomsey1 and Lois Tack, Ph.D.2
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INTRODUCTION
The need to process a greater number of convicted offender samples for DNA
analysis due to new legislation is an increasing dilemma for the forensic
community. Many states outsource their samples to private contractors to keep
up with the workload. With the availability of a new class of versatile robotic
workstation, the ability to automate a variety of testing procedures supercedes
the need for contracted processing.
The Pennsylvania State Police DNA Laboratory was tasked with processing
whole blood convicted offender samples for CODIS database entry. The
MultiPROBE® II Forensic Workstation was chosen to automate sample
processing and DNA isolation procedures. The MultiPROBE II HT EX system
with Gripper™ Integration Platform, Bar Code Reader, DPC MicroMix® 5
Shaker, Automated Heater, and PlateStak options provided the flexibility to
perform various protocols including blood spotting, DNA purification,
quantification and PCR setup.
The first automated protocol to be validated and put into daily use was paperbased archiving. This consisted of replicate spotting of convicted offender
blood onto FTA® and S & S cards for preservation, storage, and subsequent
DNA typing analysis. Blood tube samples were assigned bar codes and
spotted by the MultiPROBE II onto cards with corresponding bar codes. This
procedure utilized the workstation’s “hit picking” and liquid level sensing
features, along with bar code scanning and plate stacking functions.
Validation involved evaluating pipetting performance, sample tracking,
reproducibility, and cross-contamination.
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HARDWARE & MATERIALS
MultiPROBE II Workstation (US)
MultiPROBE II Options
AMP8E02*
AMPGRE0*
7001427
7001708
7000540
5079598
6008103
6000665
7600030
6000685
7601716
7601824*
5080165
7406626
7000008
7607651
70211890
PSS00021
70200970
MultiPROBE II HT EX w/Cmptr
Gripper Integration Platform
Left Expansion Module
ASSY, 500 uL Syringe HT
VersaTip Plus HT RoboRack Kit
4 Trough Reagent Holder, 60 mL
HT 60 mL Troughs (qty 25)
HT Reagent Trough Starter Pkg
ASSY, Filter Tip Option
175uL Clr Filter RoboRack Tips
DNA IQ System
MagneBot® 96 Magnetic
Separation Device
MagnaBot Spacer, ¼” Foam
Collection Plates, 4-pack (#)
Z3301
A9161
4
74-5563
651201
HARVARD APPARATUS 12-Ch Block
Greiner 96well V-Bottom PP Plates
95% Ethanol
100% Isopropanol
1M DTT
FTA and S&S Paper Cards
Whatman FTA Carrier Plates
EXPERIMENTAL - BLOOD SPOTTING
Auto DNA IQ Blood Protocol
The MultiPROBE II workstation has proven to be a versatile, high throughput
platform. In less than six months, over 12,000 whole blood samples have
been processed for long-term storage using FTA and S&S paper.
Simultaneously, we have carried out validation of the Promega DNA IQ
extraction procedure for DNA typing. The use of versatile automated platforms
presents a viable, in-house alternative to outsourcing. Varied procedures,
ranging from sample archiving to DNA extractions for typing, can be
automated allowing analysts to concentrate on data analysis and interpretative
activities.
Auto Blood Plating Protocol
ROBOTIC WORKSTATION
MultiPROBE II Forensic
Workstation used for
blood plating and
forensic DNA isolation
protocols. The
Integrated PlateStak
Option, used for blood
plating, is not shown.
Workstation deck
layout for blood DNA
isolation protocol. An
automated DNA IQ
Protocol using the
Promega MagnaBot®
purified DNA in 96well format from
Convicted Offender
whole blood samples.
EXPERIMENTAL – DNA PURIFICATION
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DNA DATABASE PROCESSING SCHEME
Other
Promega Part List
DC6700
V8151
Life and Analytical Sciences, Downers Grove IL
DPC MicroMix 5 Shaker Option
Auto Heater Controller Option
96U Heat Transfer Plate
ASSY, Tube BarCode HT Option
Kit, BarCode Scanner
Kit, BarCode Cassette 10-16 MM
PSS/MPII Integration Kit
PlateStak, Sgle Div Board
PlateStak Scanner
The robotic workstation was also used to automate the Promega DNA IQ™
System using whole blood samples for analysis. The goal in using this DNA
isolation protocol was to purify a consistent quantity of DNA from each sample.
Elimination of a quantification procedure is paramount in achieving the desired
throughput for processing these forensic database samples. Blood tube
samples were dispensed into 96-well plates and processed using a walk-away
60-minute protocol that required automated shaking, gripper and heater
functions. Downstream analysis of purified samples included STR-based
amplification with the PowerPlex® 16 system.
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2 PerkinElmer
Forensic DNA Laboratory, Bureau of Forensic Services, Pennsylvania State Police, Greensburg, PA
1. Load PlateStak Preferences
2. Bar Code Scan Tubes
Scan Blood Tubes in Cassettes
Auto-Create Sample List
3. Create Hits File as Well Map
4. Process Card Plates
Downstack Plate Cards with Barcode
Scan Plate Card
Merge Files, Creating New Hit List
Blood Transfer: Mix & Replicate-Spot
5. Return Plate to Stacker
Hand-Plated
File name
Robot-Plated
File name
Set #1
CPS13
CPSTR51
Set #2
CPS14
MBSTR34
Set #3
CPS15
MBSTR35
SUMMARY. Reagent blanks and
controls (9947a plus amplificationnegative) for all 6 sample sets typed
correctly. No cross-contamination was
evident for the robot-plated samples.
Robotic and manual samples typed the
same for the 16 genetic loci tested.
Sample Plating. WinPREP template directs
replicate spotting of blood samples onto FTA
and S&S cards. Up to 90 plates with cards are
loaded into PlateStak Cassette 1. Diving board
is placed at G8 on robot deck. Plate is downloaded and scanned. The card bar code is
matched with the corresponding blood tube bar
code. Blood is tip-mixed and 60 ul aliquots are
transferred in replicates of 3 to both cards.
Plate is up-stacked into Cassette 2 and the
process is continued for N# (up to 90) plates.
Present sample process time is 2.5 min/plate.
BLOOD PLATING VALIDATION
Three sets of 16 convicted offender whole
blood samples (see table) were hand-plated
onto large FTA and S&S cards as per PA
State Police DNA Database Policies &
Procedures Manual. The same sample sets
were plated onto smaller S&S and FTA (FBI)
collection cards using the MultiPROBE II.
Cuttings from both the analyst-plated and
robot-plated S&S cards (stored for ~90 days at
RT) were extracted (organic Micron Method),
quantified via a yield gel, amplified (0.5ng for
22 cycles, PowerPlex16 kit, GeneAmp 9600),
and analyzed with a ABI Prism 310 instrument
as per PA State Police DNA PCR Protocols.
Samples 2002-03282 & 2002-03287 from
MBSTR35 were also run on a ABI Prism 377.
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Auto-Scan Blood Tubes
Auto-Start Heater
Mix/Transfer Tube Blood to Plate
Add DNA IQ Resin & Lysis Buffer
Tip-Mix Sample
Incubate
Move Plate to Magnet
Aspirate Supernatant to Waste
Add Lysis Buffer Wash
Move to Shaker & AutoShake
Tip-Mix Sample
Move Plate to Magnet
Aspirate Supernatant to Waste
Add/Mix 100 uL Wash Buffer
Move to Shaker & AutoShake
Tip-Mix Sample
Move to Magnet
Aspirate Supernatant to Waste
Repeat Wash Steps x2
Air Dry 4 min
Add 100 uL Elution Buffer
Move to Heater Tile on Shaker
Heat at 65C for 3 min
Shake & Heat at 65C for 5 min
Move to Magnet
Transfer DNA to Collection Plate
Forensic DNA Isolation. A WinPREP DNA IQ
template, created to process forensic blood samples, is
used to prepare DNA in 96-well format. Blood is
transferred (using 8-tips per transfer step) from tubes to
a 96-well plate using the robot’s VariSpan feature. The
template then directs an automated DNA IQ purification
protocol. The DNA IQ resin, designed to capture fixed
amounts of DNA, is added to cell lysates to capture
genomic and mitochondrial DNA. After ethanol
washing, bound DNA is eluted using an automated
heating and shaking step. Purified DNA is transferred
to a clean polypropylene 96-well plate.
Protocol
processing time is 90-120 min for 96 samples. Note:
the DPC Shaker with heater tile was placed “off-deck”
to increase the working deck space for the pipettor.
DNA EXTRACTION VALIDATION
ROBOT SET 1. Saturation study to optimize extraction
program and chemical preparation methods. Thirteen
blood sets (260 convicted offender whole bloods; 30
separate extractions) were analyzed altering various
parameters to optimize yields and consistency for the
DNA IQ procedure using the MultiPROBE II robot. See
Optimization Table below for results.
PARAMETER TESTED
OPTIMIZATION SOLUTION
Pellet loss - “popping” during aspiration
All aspiration heights evaluated. Tips adjusted.
Volume blood extracted
10, 15, 20, 30, 40 uL
Amount of resin addded
3.5 uL/well (1/2 rx) vs 7 uL/well (full rx)
Shaker vs disposable tip-mixing
Mixing lysate + resin following blood addition with
conductive tips, clear tips, shaker, or tip mix w/ shaker
Lysis Buffer + DTT preparation
Lysis Buffer w/ DTT tested, prepared fresh to weeks old
Elution Heat Transfer Blocks
96U Silver MPII prototype, Promega PPN Z3271,
96U MPII Blue Heat Transfer Block PPN 5080165
Removal of final elution color
Add “pellet buster” steps
Blood transfer: tubes to plate
200 uL tips w/ blood mixing vs 25uL tips w/o blood mixing
*incorporated as final parameter
ROBOT SET 2. Five separate extractions were carried out using the DNA IQ system and the robot with
the same set of 32 whole blood convicted offender samples. The results on varying volumes of DNA IQ
resin and whole blood are shown below and are part of the optimization table shown above.
Plate Name
SAT 14.10F
SAT 14.15F
SAT 14.20F
SAT 14.10H
SAT 14.15H
Resin, uL
7
7
7
3.5
3.5
Blood, uL
10
15
20
10
15
Avg Yield, ug/uL
.658
.689
.863
.360
.406
% Desired Yield Range*
97
91
88
75
66
*For sets utilizing 7 uL/sample of beads, desired yield range is 0.5 ng/uL to 1 ng/uL
For sets utilizing 3.5 uL/sample of beads, desired yield range is 0.25 ng/uL to 0.5 ng/uL
Summary. A blood volume of 15 uL with a resin volume of 7 uL/well gave the best results.
Present and Future Laboratory
Sample Processing Schemes. The
upper panel shows our current
laboratory database process. The
MultiPROBE
II
workstation
is
presently used to carry out automated
bar code scanning of blood tubes,
tracking of matching bar code-labeled
paper filters, blood plating, sample
reformatting, and DNA isolation from blood samples. In the near future, the lower panel shows
additional applications to be automated using this workstation. They include DNA extraction from
blood stains, DNA normalization, and PCR set-up.
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SUMMARY
The MultiPROBE II Workstation offers the following features for forensic database
sample processing, DNA purification and analysis:
ƒ Automated protocol carries out barcode scanning, matching and spotting steps
needed for convicted offender database sample archival.
ƒ Integrated BarCode Tube Reader and PlateStak with top-read card scanner
allowed processing of 90 samples 12,000 samples in less than 6 months.
Processing time per plate was 1 to 2.5 min, depending on blood tube size.
ƒ Walk-away protocol carries out all steps needed for the automated paramagnetic
particle-based DNA IQ System. Optimized yields were 97% of expected and
ranged from 0.7-0.9 ug/uL.
ƒ Blood is transferred from tubes to plates using MultiPROBE ‘s VariSpan® feature.
ƒ Integrated gripper moves sample microplate between the magnet and shaker
during wash steps. Integrated shaker used for automated cell lysis and DNA
extraction steps; increases throughput and improves DNA yields.
ƒ DNA IQ processing time is between 90-120 min for a 96-well sample plate.
ƒ Optimized forensic protocol prepares DNA suitable for automated PCR analysis
using PowerPlex amplification kits.
ƒ The workstation can be used to carry out other forensic nucleic acid purification
applications including QIAGEN, Machery Nagel, and AGOWA kits.
ƒ Other MultiPROBE II accessories allow workstation to carry out automated
protocols such as DNA quantification, DNA normalization, and PCR set up.
References
1. Krenke, BE, A Tereba, SJ Anderson, E Buel, S Culhane, CJ Finis, CS Tomsey, et al. 2002.
Validation of a 16-locus fluorescent multiplex system. J Forensic Sci 47(4): 773-785.
2. Tack, L & T Grunst. 2002. Automated Forensic DNA Isolation from Blood using Promega’s
DNAS IQ System and the MultiPROBE II Forensics DNA Workstation. Poster: SAFS Fall Annual
Meeting, Sept 30-Oct 3, 2002, Bioloxi MS.
3. Tack, L. 2003. PerkinElmer’s MultiPROBE II Forensic Workstation. Application Note: AAFS 55th
Annual Meeting, Feb 18-21, 2003, Chicago IL .
4. Promega Technical Bulletin #TB297: “DNA IQ System – Database Protocol”. 6/02
5. Comey, C.T., Koons, B.W., Presley, K.W., Smerick, J.B., et al. 1994. DNA extraction strategies
for amplified fragment length polymorphism analysis. J For Sci 39 (5): 1254-1269.
6. Promega Technical Bulletins #TMD012 and TMD006: "PowerPlex 16 System", 10/02 and
“GenePrint® Fluorescent STR Systems”. 6/02.
Acknowledgements
We thank Peng Li, Bruce Tyley, Jack Judson and Karen Michalski at PerkinElmer for invaluable help in
developing hardware and software for this MultiPROBE II Workstation. Andrew Raneri helped with
the PlateStak hardware & software operation. Angelina Croston carried out most slot blot QA assays.