A High-Throughput Device for Processing Samples on Solid Supports

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 formula:

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.

Device components

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 being developed.

Reference

  1. 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.

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