Crime statistics now support the effectiveness of
aggressively analyzing breaking-and-entering cases
and developing genotype databases of these felons.
Unfortunately, this approach has created an ever-expanding
workload and increasing backlogs.
Over the last few years, automation has played an
important and expanding role in handling the
increased work, but the up-front processing of these
samples still creates a bottleneck. Most casework,
reference, and database samples are on solid supports
that do not lend themselves to robotic manipulation.
FTA® cards (Promega Corp., Madison, WI) have
provided one solution for reference and database
samples. However, because of the high DNA binding
capacity, they typically give poor genotype profiles
unless the DNA is removed.
Although automated methods exist for buccal
swabs, they can give variable results and are prone
to clogged pipet tips. Previously, casework samples
needed to be processed individually to separate the
solid support from the eluted DNA solution. This
bottleneck has been solved through the development
of the Slicprep™ 96 device (cat. no. V1391,
Promega Corp.). The product allows the simultaneous
centrifugation of 96 samples and is designed so
that both the digestion or lysis and centrifugation
can be performed in the same device. The portable,
lightweight F3K tabletop centrifuge (FIBERLite
Centrifuge Inc., Santa Clara, CA, Figure 1) was
used for testing. The centrifuge’s H-3S horizontal
rotor features modified plate carriers specifically
designed for use with the unit’s 96 Spin Baskets. The
U-shaped collar of the baskets is placed in the digestion
or spin position in the 96-deep-well format plates.
Figure 1 - F3K portable tabletop centrifuge with plate carrier.
Tests were performed to simulate digestion,
lysis, and centrifugation of 96 samples
in the same device. Samples such as
cotton swabs, blood punches, or pieces of
clothing can be inserted in the spin basket
to ensure good removal of DNA and cells
from the solid support during criminal
investigations. The digestion and centrifugation
methods followed are listed
below and have been reported previously.1
The F3K centrifuge has a fixed speed and
variable run time setting. The maximum
speed of the rotor is 3000 rpm, which generates
1600 × g. When fully loaded, the
rotor takes 2.0 min to achieve maximum
speed. The calculated run time for the
required study is obtained from the following
Calculated run time = reported g-force ×
reported run time/maximum g-force of rotor
where reported g-force = 1500 × g,
reported run time = 5 min, maximum force
= 1600 × g, calculated run time = 4 min + 2
min acceleration time, and set run time = 6.0 min.
Figure 2 - Components of the Slicprep 96 device: a) 96 Spin
Basket; b) U-shaped collar; and c) 2.2-mL, 96-deep-well plate.
The Slicprep 96 device consists of three components:
the 96 Spin Basket, U-shaped collar, and 2.2-mL solution
(ABgene, Rochester, NY), 96-deep-well plate
(Figure 2a–c). The seven holes in the rounded bottom of
the baskets ensure good removal of DNA and cells from
the solid support. Samples such as cotton swabs, blood
punches, or pieces of clothing are inserted into the baskets,
which are big enough to accept an entire dried cotton
swab. In the digestion position, the spin basket is
fully inserted into the deep-well plate, allowing space for
approx. 165 μL of solution below the basket in each well
(Figure3a). After the incubation, the baskets are raised
approx. 1 cm (the spin position, Figure 3b) to create
space for an additional 500 μL of solution.
Materials and methods
With the device in the digestion position, samples
were placed in the baskets. For reference samples, 400
μL of DNA IQ™ lysis buffer (Promega Corp.) was
added and the device was sealed with a foil seal and
heated in a 70 °C water bath for 1 hr. For touch samples,
400 μL of a 1.8 μg/μL proteinase K solution was
added, and samples were incubated in a 56 °C oven
for 1 hr. After incubation, the device was removed
from the water bath or oven and the U-shaped collar
was inserted. The device was then centrifuged for 6
min in the centrifuge with the horizontal rotor.
Figure 3 - Operational modes of the device. a) Digestion
position: The spin basket is fully inserted into the 96-deep-well
plate to allow incubation of solid supports with digestion or lysis
buffer. b) Spin position: The U-shaped collar is inserted
between the 96-deep-well plate and spin basket. This raises the
basket and allows an additional 500 μL below the basket.
The collar and baskets were then discarded, leaving
the DNA-containing solution in the deep-well plate.
The plate was placed on a Biomek® 2000 workstation
(Beckman Coulter, Fullerton, CA), and a Bio
Works™ method (Bio Works Inc., Baltimore, MD)
directed DNA purification using the DNA IQ system.
DNA was eluted in 100 μL (reference samples) or 40 μL (touch samples) of TE-4 buffer (10 mM Tris
[pH 8.0], 0.1 mM EDTA). Finally, a total of 1 μL of
the eluted DNA or 10 μL of eluted control (blank)
DNA was amplified with the PowerPlex® 16 system
(PowerPlex, West Conshohocken, PA) and analyzed
on a PRISM® 3100 Genetic Analyzer (Applied
Biosystems [ABI], Foster City, CA).
Processing FTA card punches and buccal swabs
The Slicprep 96 device is well suited for high-throughput
extraction of DNA from database samples
such as blood cards and buccal swabs. One 4-mm-diam or three 2-mm-diam FTA punches containing
blood, and whole cotton and paper (CEP)
buccal swabs, were processed as described in the
“Materials and methods” section using 400 μL of
DNA IQ lysis buffer (500 μL for paper swabs).
Automation with the device
The device is designed to provide higher throughput
than individual spin baskets for up-front processing
of samples on solid supports. For database and reference
samples where the sample is soaked in DNA
IQ lysis buffer, the extracted fractions in the deep-well
plate can be processed using existing methods.
Touch samples are best processed with up to 500 μL
of proteinase K digestion solution, which must be
diluted with two volumes of DNA IQ lysis buffer
before DNA purification. The resulting large volumes
are not efficiently processed by most workstations.
However, Dr. Susan Greenspoon (Virginia
Division of Forensic Sciences, Richmond, VA) has
collaborated with Promega Corp. to develop a
Biomek 2000 method that can handle aqueous sample
volumes up to 0.5 mL as efficiently as the manual
DNA IQ system or phenol:chloroform purification.
A method to incorporate large volumes is
- Tereba, A.; Krueger, J.; Olson, R.; Mandrekar, P.; McLaren, B. Profiles in DNA. Promega Corp.: Madison, WI, Sept 2005.
Dr. Griffith is Director of Research, FIBERLite Centrifuge Inc.,
422 Aldo Ave., Santa Clara, CA 95054, U.S.A.; tel.: 408-988-1103; fax: 408-988-1196; e-mail: MGriffith@piramoon.com.