STR amplification of DNA from buccal and blood samples stored long-term on Whatman FTA cards

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1 GE Healthcare Life Sciences Application note AA STR amplification of DNA from buccal and blood samples stored long-term on Whatman FTA cards FTA technology In this study we successfully amplified DNA from 22-year-old blood and 12-year-old buccal samples stored on FTA cards at ambient temperature. Several commercially available kits for short tandem repeat (STR) amplification were used, including kits for both standard ( punch-in ) and direct STR amplification. Interpretable STR profiles were generated from all blood and buccal samples using the Applied Biosystems AmpFlSTR Profiler Plus, AmpFlSTR COfiler, and AmpFlSTR Identifiler Direct PCR Amplification Kits from Life Technologies. These data are relevant to forensic laboratories because they demonstrate in functional terms the ability of FTA to overcome environmental and in vivo factors, both of which can harm DNA during long-term storage. Introduction Whatman FTA technology is used globally in forensic labs for long-term, ambient temperature archiving of single-source DNA samples. These samples are often tested at the time of collection, and then stored. However, retesting of these samples can occur months to years later upon request for verification purposes. This study was designed to use STR analysis to demonstrate that Whatman FTA cards are effective matrices for sample collection and long-term, room-temperature archiving for a decade or more. Whatman FTA cards from GE Healthcare Life Sciences are chemically coated matrices that have been shown to preserve DNA more efficiently than untreated matrices. Cells in biological samples deposited on FTA cards lyse on contact and DNA is captured on the card. DNA is protected from environmental and enzymatic damage by the FTA chemicals embedded in the card. Whatman FTA technology is available in multiple off-the-shelf (Fig 1A) and custom configurations (Fig 1B) that are designed to fit into your laboratory s workflows.

2 Fig 1A. Whatman FTA cards, Indicating FTA cards, and EasiCollect device. Fig 1B. Example of Custom multifunctional kit (blood, buccal cells, or saliva) and Custom FTA cards AA

3 Materials and methods Sample collection Blood samples were spotted onto FTA Cards (GE Healthcare) in Buccal samples were collected onto Indicating FTA cards (GE Healthcare) in All samples were stored at ambient temperature inside multi-barrier pouches with desiccant. Samples were stored in a fireproof safe. Sample preparation for PCR amplification and analysis Sample processing for standard, punch-in STR amplification Blood and buccal samples on FTA cards were prepared for short tandem repeat (STR) amplification using the following modified FTA punch-in protocol (provided with permission from Dr. Arthur Eisenberg): 1. A 1.2 mm punch was taken from each blood or buccal sample and placed in a 200 µl amplification tube. 2. FTA Purification Reagent (200 µl; GE Healthcare) containing 60 µg/ml Proteinase K was added to the tube, which was then incubated at 65 C for 30 min. 3. The contents were mixed by pipetting the solution up and down three times, and the wash solution was aspirated from the FTA punch and discarded. 4. FTA Purification Reagent (200 µl) without Proteinase K was added, and the tube was incubated at room temperature for 5 min. 5. The contents were mixed by pipetting the solution up and down three times, and the wash solution was aspirated and discarded. 6. Step 5 was repeated two times for a total of three washes. 7. TE -1 buffer (10 mm Tris-HCl, 0.1 mm EDTA, ph 8.0; 250 µl) was added to the FTA punch, and the tube was incubated at room temperature for 5 min. 8. The contents were gently mixed by pipetting the buffer up and down three times, and the buffer was removed and discarded. 9. Step 8 was repeated three times for a total of four washes. 10. A pipette was used to remove as much of the buffer as possible from the tube and sample punch. The tube was incubated at 65 C for 30 min to dry the punch prior to standard, punch-in PCR amplification. For each amplification reaction, one dried FTA punch was placed in a 0.2 ml tube. Master mix (6 µl containing primer, reaction mix, and AmpliTaq Gold DNA polymerase) from either the Profiler Plus or the COfiler kit and 4 µl of water were added to each tube. The processed FTA punches and the kit-supplied controls were amplified using a 96-Well GeneAmp PCR System The manufacturer s recommended thermal cycling conditions were followed for 24 cycles (blood and buccal on FTA) and 28 cycles (controls).* Sample processing for direct STR amplification A 1.2 mm punch was taken from each blood or buccal sample and was added to 25 µl of Master Mix from an Identifiler Direct PCR Amplification Kit. Thermal cycling was performed on a GeneAmp PCR System 9700 following the manufacturer s protocol for 27 cycles (blood and buccal on FTA and controls).* Capillary electrophoresis setup and analysis Capillary electrophoresis (CE) and reaction plate setup were performed according to the manufacturer s instructions. All samples were run on an Applied Biosystems 3130xl Genetic Analyzer using recommended methods supplied for each chemistry type. The only modification to the recommended 3130xl running conditions was a 15 sec injection time for both the blood and buccal samples processed using the standard, punch-in method on FTA. Data analysis was performed using GeneMapper ID Software v3.2 with a peak amplitude threshold of 50 relative fluorescence units (RFU) for allele calling.* Acceptance criteria For each sample, the profiles generated from the Profiler Plus and COfiler STR reactions were compared to the profiles that were generated when the same samples were 14 (blood) and 5 (buccal) years old. In addition to concordance, the following criteria were applied: All controls should provide the expected results as outlined in the kit instructions. Heterozygous Peak Height Ratios should exceed 50%. Heterozygous Peak Height Threshold set at 100 RFU. Homozygous Peak Height Threshold set at 200 RFU. * All materials used in this section were purchased from Life Technologies Set in GeneMapper ID Analysis Method as minimum peak height process quality values threshold (PQV) AA 3

4 Results and discussion AmpFlSTR Profiler Plus and COfiler PCR Amplification Kits (standard, punch-in method) When used with both kits, the 22-year-old blood and 12-year-old buccal samples on Whatman FTA and Indicating FTA cards produced full profiles that met all acceptance criteria listed in Materials and methods. Buccal samples (12 years old) amplified with the Profiler Plus kit (Fig 2A) and COfiler kit (Fig 2B) yielded full STR profiles with an average peak height of 266 and 295 RFU, respectively. Both profiles showed concordance with the data that had been generated from FTA cards in 2004 (data not shown). Fig 2. STR profiles of a 12-year-old buccal sample collected on Indicating FTA cards. STR amplification was performed using the following Applied Biosystems kits: A) Sample BC-PP, AmpFlSTR Profiler Plus PCR Amplification Kit; and B) Sample BC-CO, AmpFlSTR COfiler PCR Amplification Kit. A 15 sec injection time was used on the AB 3130xl Genetic Analyzer. A) Sample BC-PP B) Sample BC-CO AA

5 Blood samples (22 years old) amplified with the Profiler Plus kit (Fig 3A) and COfiler kit (Fig 3B) yielded full STR profiles with average peak height of 1595 and 1076 RFU, respectively. Both profiles showed concordance with data generated from FTA cards in 2004 (data not shown). Fig 3. STR profiles of a 22-year-old blood sample collected on FTA cards. STR amplification was performed using the following Applied Biosystems kits: A) Sample BL-PP, AmpFlSTR Profiler Plus PCR Amplification Kit; and B) Sample BL-CO, AmpFlSTR COfiler PCR Amplification Kit. A 15 sec injection time was used on the AB 3130xl Genetic Analyzer. A) Sample BL-PP B) Sample BL-CO AA 5

6 AmpFlSTR Identifiler Direct PCR Amplification Kit (direct amplification) The 22-year-old blood and 12-year-old buccal samples produced full profiles that met all of the acceptance criteria in Materials and methods. The average peak height was 3824 RFU for blood and 2312 RFU for buccal samples. Both the blood and buccal samples processed using Identifiler Direct showed concordance with the corresponding alleles in Profiler Plus and COfiler (data not shown). Figure 4 shows an STR profile using Identifiler Direct for amplification of DNA in buccal (Fig 4A) or blood (Fig 4B) on Indicating FTA and FTA, respectively. Fig 4. STR profiles generated using AmpFlSTR Identifiler Direct PCR Amplification Kit to amplify DNA from A) Sample BC-ID, a 12-year-old buccal sample collected on Indicating FTA; and B) Sample BL-ID, a 22-year-old blood sample collected on FTA Cards. A) Sample BC-ID B) Sample BL-ID AA

7 Conclusions This study demonstrates that Whatman FTA technology is effective for archiving DNA from both buccal and blood samples for more than a decade. The data showed that commercially available kits for punch-in and direct STR amplification were able to produce full, database-uploadable profiles from the 22-year-old blood and 12-year-old buccal samples on both FTA and Indicating FTA matrices. The modified FTA Reagent/Proteinase K punch-in protocol was able to generate full profiles using the AmpFlSTR Profiler Plus and COfiler PCR Amplification Kits; however, this method was not as efficient as direct amplification using the AmpFl- STR Identifiler Direct kit. The direct amplification method with FTA was faster, eliminating about 1.5 h of sample processing time. In addition, it produced much higher peak heights than punch-in amplification using either the Profiler Plus or COfiler kits. The buccal sample on Indicating FTA showed lower peak heights overall for both direct and standard, punch in amplification methods compared to the same sample on FTA Classic Cards (data not shown). However, this decrease in performance may be attributed to less sample being present on the Indicating card; extensive previous rounds of testing left only the edges of the sample available for punching AA 7

8 GE, imagination at work, and GE monogram are trademarks of General Electric Company. EasiCollect, FTA, and Whatman are trademarks of GE Healthcare companies. For local office contact information, visit: GE Healthcare UK Limited Amersham Place, Little Chalfont Buckinghamshire, HP7 9NA UK AmpFlSTR, Applied Biosystems, COfiler, GeneAmp, GeneMapper, Identifiler, and Profiler Plus are trademarks of Life Technologies. AmpliTaq Gold is a trademark of Roche Molecular Systems, Inc General Electric Company All rights reserved. First published Dec All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare Bio-Sciences AB Björkgatan Uppsala Sweden GE Healthcare Europe, GmbH Munzinger Strasse 5 D Freiburg Germany GE Healthcare Bio-Sciences Corp. 800 Centennial Avenue, P.O. Box 1327 Piscataway, NJ USA GE Healthcare Bio-Sciences KK Sanken Bldg., Hyakunincho Shinjuku-ku, Tokyo Japan AA 11/2011