- Email: [email protected]
Automated Forensic DNA Purification Optimized for FTA Card Punches and Identifiler STR-based PCR Analysis
Clin Lab Med 27 (2007) 183–191
Automated Forensic DNA Purification Optimized for FTA Card Punches and Identifiler STR-based PCR Analysis Lois C. Tack, PhDa,*, Michelle Thomas, MSb, Karl Reich, PhDb a
PerkinElmer Life and Analytical Sciences, Liquid Handling Applications Group, 2200 Warrenville Road, Downers Grove, IL 60515, USA b Independent Forensics of Illinois, 1960 Springer Drive, Hillside, IL 60148, USA
Utilizing automation to reduce sample backlogs is the paramount objective for most crime labs today. An important part of an automation approach involves identifying the most rapid protocol that still provides an accurate STR profile for a given sample type. Automating the Whatman FTA DNA Purification Kit (Whatman Inc., Clifton, New Jersey) is an important part of this application strategy as it can maximize processing throughput for database and reference casework sample types. Purification of forensic samples immobilized on FTA chemically treated paper punches involves simple washing of the paper discs followed by drying. PCR amplification of the DNA is carried out in situ, followed by STR analysis. The method is rapid, easy to automate, efficient, robust, and economical. Thus, it is an ideal automated application for processing database and reference casework samples when collected as blood spots or fresh buccal swipes on FTA paper.
FTA procedure overview FTA technology allows for the collection, transport, and stable storage at room temperature of nucleic acid samples from a wide variety of sample types [1,2,4]. These include blood, buccal swipes, cultured cells, tissue, and microorganisms. It is widely used in human forensic analysis as DNA This article was originally published in JALA; August 2005. * Corresponding author. E-mail address: [email protected] (L.C. Tack). 0272-2712/07/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cll.2006.12.009 labmed.theclinics.com
184
TACK
et al
processing can be carried out months or years later, making this an ideal format for sample archiving. An FTA wash procedure to prepare samples for STR analysis suitable for Combined DNA Index System (CODIS) database entry has been described [3]. However, validation was carried out using the robust, well-characterized ABI AmpFlSTR Profiler Plus PCR Amplification Kit with its simpler three-color 9 STR loci profile. The newer AmpFlSTR Identifiler PCR Amplification Kit contains 15 STR loci, which can be more temperamental and require changes in the DNA isolation method to optimize its more complex five-color profile. To automate the FTA wash method for use with the Identifiler system, we evaluated various reagents (including the Whatman FTA Purification Kit WB120204), washing protocols, mixing routines, drying conditions, punch size, and STR reaction conditions. This document describes the conditions that provided the best results. The protocol steps are outlined in Fig. 1. System overview The goal is to develop a fully automated method to prepare FTA DNA templates in 96-well plates suitable for multiplex STR analysis using the Identifiler kit. The automated method utilizes a single PCR plate as both the sample source plate and final destination plate. The purified DNA in the PCR plate is ready for direct STR amplification and standard forensic genotyping analysis using conventional capillary electrophoresis (CE) systems. The walk-away procedure uses commercially available reagents and is optimized for tip and reagent usage and other parameters. The system consists of a MultiPROBE II HT EX Workstation with several integrated options. Key hardware and software features are (1) VersaTip RoboRack Plus Option. This allows automated tip mode switching, so that one can change, in the same step, between using eight fixed tips (reagent dispensing) and eight disposable tips (mixing and waste removal steps). The same disposable filter tip is also used to carry out a preaspirate mix step for three cycles before waste transfer. (2) Software-adjusted syringe motors. All waste aspiration steps used syringe motor speeds set at 25% of normal to minimize foaming of the kit reagent and to eliminate aspiration of the paper disc. (3) MPII Automated Heater Controller and PCR96 Heat Transfer Block. This softwarecontrolled device can heat up to four heater tiles independently and is used for drying washed FTA punches (Fig. 2). The off-deck controller preserves deck space. The heater tile with a PCR plate and PCR96 heat transfer block assembly is placed on the main deck. (4) 175 mL RoboRack Filter Disposable Tips. Barrier tips prevent cross-contamination when processing multiple samples. Fig. 3 shows the deck layout as it appears in the WinPREP software test outline. Note the location of the heater tile assembly. The workstation also included the Gripper Integration Platform and DPC MicroMix 5 Shaker
AUTOMATED FORENSIC DNA PURIFICATION
185
Fig. 1. Automated FTA DNA purification procedure.
(Diagnostics Products Corporation, Los Angeles, California) Options. Neither is required for a one-plate protocol. Optimized FTA protocol for identifiler analysis Four reference bloodstained FTA card samples of known genotypes were obtained from the FBI and processed manually in 1.5-mL-conical tubes. To optimize for analysis with the Identifiler Kit [5,6], three different DNA purification protocols and various STR PCR setup methods were compared.
186
TACK
et al
Fig. 2. Automated MultiPROBE II Heater Controller with quad heater tiles.
Variables tested are shown below. All samples were analyzed on either an ABI PRISM 310 or 3100 Genetic Analyzer using GeneScan and Genotyper software. The following methods were tested: (1) The Whatman 2003 FTA application note method, (2) the ABI-modified FTA method (ABI customer support), and (3) the NaOH (10 mM) wash method [2]. The variables tested were punch sizes (1.2 mm, 2 mm); wash conditions (FTA buffer, 10 mM NaOH, TE-1, water); drying (air-dry or 50 C heater);
Fig. 3. MultiPROBE II FTA DNA purification protocol deck layout.
AUTOMATED FORENSIC DNA PURIFICATION
187
Fig. 4. Genotyper electropherogram for reference sample 4.
STR kits (Identifiler (15 STR loci þ amelogenin), Profiler Plus (9 STR loci þ amelogenin) PCR reaction volumes (15 mL (undiluted), 25 mL (þ 10 mL TE-1) and PCR cycles (28 or 24). The ABI-modified FTA method 2, when used with Identifiler amplification, gave the best results. The overall processing scheme used with method 2 is as follows: Prepare 1.2 mm FTA punches using a Harris Micro Punch in PCR microfuge tubes. Wash twice with 180 mL FTA Purification Reagent: vortex, incubate 5 minutes, aspirate supernatant. Wash once with 180 mL TE-1: vortex, incubate 5 minutes, aspirate supernatant. Wash once with H2O: vortex, incubate 5 minutes, aspirate supernatant. Dry at 50 C for 10–15 minutes. Add 15 mL Identifiler master mix (no water added to sample disc). Amplify 24 cycles using kit recommended cycling parameters in an Applied Biosystems GeneAmp PCR System 2700 (Applera Corporation, Foster City, California). Add size standards. Denature and load 1 mL on CE system. All samples processed showed sharp well-resolved peaks with high sensitivity (w 3000–8000 RU). All 15 STR alleles were easily called using Genotyper software. Optimum conditions included partial volume PCR
188
TACK
et al
Fig. 5. (A) Checkerboard plate format used for cross-contamination analysis; (B) Cross-contamination assay results. Yes ¼ correct STR profile; bl ¼ blank profile with no STR peaks but standards are present.
conditions (15 mL master mix, undiluted) with PCR cycle number n ¼ 24. Note: samples processed using NaOH wash method 3 showed well-resolved peaks with all 15 STR alleles called correctly, but with much reduced peak heights compared to method 2 (not shown). Automated FTA DNA purification protocol Reference sample punches were processed using the MultiPROBE II workstation and a WinPREP template created using method 2 parameters (see Fig. 1). Discs were placed in a 96-well PCR plate on the heater tile assembly located on the main deck (see Fig. 3). After washing, the sample plate was automatically heated to 50 C. All waste removal steps used 25% of normal aspirate motor speeds to minimize disk contact and avoid aspirating punches during the wash steps. Reagents were replicate-dispensed using fixed tip mode at 101% well height to maximize throughput without contamination. Reagent waste was removed using disposable filter tips. The processing time is w 30 minutes for 96 samples, not including a 20 minutes
AUTOMATED FORENSIC DNA PURIFICATION
189
automated drying step. About 20 minutes was dedicated to soak time for the several washes used. Purified DNA is ready for immediate STR analysis. Identifiler master mix was added to each well and samples were amplified and analyzed on an ABI Prism 3100 Genetic Analyzer. A typical electropherogram for a purified punch sample is shown in Fig. 4 and displays the 15 STR loci and the Amelogenin locus expected. Each dye panel is shown separately; the orange LIZ size standard peaks are shown in the bottom panel. Potential cross-contamination of samples was evaluated using a checkerboard pattern of reference punches and empty wells in a PCR plate as shown in Fig. 5A. Pink wells each contained a different known bloodstained punch. Remaining wells are empty blanks. After processing using the MultiPROBE II workstation, both sample and blank wells were analyzed using Identifiler analysis. The STR analysis of FTA reference sample 2 processed in well C3 is shown in Fig. 6A. STR analysis results for blank well B3 are
Fig. 6. (A) FTA sample STR analysis results for well C3; (B) blank sample STR analysis results for well B3.
190
TACK
et al
shown in Fig. 6B. B3 is adjacent to well C3. The 5-dye set for B3 is displayed as a single panel. Only the LIZ dye standard peaks added to each sample before CE analysis are present, as expected. The results for one experiment are shown in Fig. 5B. No evidence of cross-contamination was found in any sample analyzed. The reference samples all gave the correct STR profiles. No STR peaks were observed in any blank wells. Peak height, stutter percentages, and overall quality of electropherograms using Identifiler and Profiler Plus [7] kits were identical or better than manual sample purification. Fig. 7 shows a summary of the Identifiler alleles, correctly called for four bloodstained FTA reference samples purified using the MultiPROBE II Forensic Workstation and reagents from the Whatman FTA Purification Kit.
Summary We describe a rapid, simple, and reliable procedure for automated processing of 1.2 mm punches from FTA paper stains for PCR-based STR analysis. The robotic procedure uses Whatman FTA Purification Kit reagents and was optimized for use with Identifiler STR typing. Comparable results were obtained with Profiler Plus kits. All DNA STR profiles were compared against results obtained using traditional extraction techniques and were congruent at all loci tested. The protocol is easily scalable. The 30 min/plate run time is decreased if multiple plates are processed at once. Punches 1.2 mm in size provide ample DNA for multiple STR analysis runs. The larger 2 mm bloodstained punches did not give as good results using the Identifiler kit. Fully automated drying using software-directed temperature control is available with the heater option. The Gripper Integration Platform option is not required for processing small plate numbers but may
Fig. 7. Reference sample STR typing results.
AUTOMATED FORENSIC DNA PURIFICATION
191
be used to move plates to the off-deck heater or for repopulating the deck if processing large plate batches. The same robot system can be used to automate other forensic sample protocols. This includes DNA quantification, normalization, dilution, PCR setup assays, and the Promega DNA IQ System Kit for casework sample DNA isolation.
Acknowledgments We gratefully thank the following: P. W. Boonlayangoor, Ph.D., Laboratory Director, Independent Forensics of Illinois, Lombard, IL; Rob McPheeters, Automations Specialist, Genetic ID, Whatman, Inc., Clifton, NJ; Xavier Aranda, DNA Identity Lab, University of North Texas, Fort Worth, TX; and ABI Technical Support at Applied Biosystems Inc., Foster City, CA.
References [1] Whatman Inc. FTA Protocols: Collect, Transport, Archive and Access Nucleic Acids - All at Room Temperature; WB120047; Copyright Whatman Inc., 2002. Available at: www. whatman.com. [2] Smith LM, Burgoyne LA. Collecting, archiving and processing DNA from wildlife samples using FTA databasing paper. BMC Ecology 2004;4:4–15. [3] Whatman Inc. Preparation of Isolated DNA from FTA Cards Using an Automated Liquid Handling System; Application Note 51609; Copyright Whatman Inc., 2003. [4] Smith MA, Igoe F, Pierce K, et al. FTA Technology – New Advances for the Collection, Shipment, Archiving and Processing of Biological Samples for Human Identification. Presented at the 13th International Symposium on Human Identification, Phoenix AZ, 2002; Poster Abstract. [5] Applied Biosystems. AmpFlSTR Identifiler PCR Amplification Kit; Human Identification Publication 112PB02-03; Copyright Applied Biosystems 2002. [6] Applied Biosystems. AmpFlSTR Identifiler PCR Amplification Kit User’s Manual; Product Manual PPN 4323291; Copyright Applied Biosystems 2002. [7] Applied Biosystems. AmpFlSTR Profiler PCR Amplification Kit; Human Identification Publication 112PB02-01; Copyright Applied Biosystems 2002.
Automated Forensic DNA Purification Optimized for FTA Card Punches and Identifiler STR-based PCR Analysis Lois C. Tack, PhDa,*, Michelle Thomas, MSb, Karl Reich, PhDb a
PerkinElmer Life and Analytical Sciences, Liquid Handling Applications Group, 2200 Warrenville Road, Downers Grove, IL 60515, USA b Independent Forensics of Illinois, 1960 Springer Drive, Hillside, IL 60148, USA
Utilizing automation to reduce sample backlogs is the paramount objective for most crime labs today. An important part of an automation approach involves identifying the most rapid protocol that still provides an accurate STR profile for a given sample type. Automating the Whatman FTA DNA Purification Kit (Whatman Inc., Clifton, New Jersey) is an important part of this application strategy as it can maximize processing throughput for database and reference casework sample types. Purification of forensic samples immobilized on FTA chemically treated paper punches involves simple washing of the paper discs followed by drying. PCR amplification of the DNA is carried out in situ, followed by STR analysis. The method is rapid, easy to automate, efficient, robust, and economical. Thus, it is an ideal automated application for processing database and reference casework samples when collected as blood spots or fresh buccal swipes on FTA paper.
FTA procedure overview FTA technology allows for the collection, transport, and stable storage at room temperature of nucleic acid samples from a wide variety of sample types [1,2,4]. These include blood, buccal swipes, cultured cells, tissue, and microorganisms. It is widely used in human forensic analysis as DNA This article was originally published in JALA; August 2005. * Corresponding author. E-mail address: [email protected] (L.C. Tack). 0272-2712/07/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cll.2006.12.009 labmed.theclinics.com
184
TACK
et al
processing can be carried out months or years later, making this an ideal format for sample archiving. An FTA wash procedure to prepare samples for STR analysis suitable for Combined DNA Index System (CODIS) database entry has been described [3]. However, validation was carried out using the robust, well-characterized ABI AmpFlSTR Profiler Plus PCR Amplification Kit with its simpler three-color 9 STR loci profile. The newer AmpFlSTR Identifiler PCR Amplification Kit contains 15 STR loci, which can be more temperamental and require changes in the DNA isolation method to optimize its more complex five-color profile. To automate the FTA wash method for use with the Identifiler system, we evaluated various reagents (including the Whatman FTA Purification Kit WB120204), washing protocols, mixing routines, drying conditions, punch size, and STR reaction conditions. This document describes the conditions that provided the best results. The protocol steps are outlined in Fig. 1. System overview The goal is to develop a fully automated method to prepare FTA DNA templates in 96-well plates suitable for multiplex STR analysis using the Identifiler kit. The automated method utilizes a single PCR plate as both the sample source plate and final destination plate. The purified DNA in the PCR plate is ready for direct STR amplification and standard forensic genotyping analysis using conventional capillary electrophoresis (CE) systems. The walk-away procedure uses commercially available reagents and is optimized for tip and reagent usage and other parameters. The system consists of a MultiPROBE II HT EX Workstation with several integrated options. Key hardware and software features are (1) VersaTip RoboRack Plus Option. This allows automated tip mode switching, so that one can change, in the same step, between using eight fixed tips (reagent dispensing) and eight disposable tips (mixing and waste removal steps). The same disposable filter tip is also used to carry out a preaspirate mix step for three cycles before waste transfer. (2) Software-adjusted syringe motors. All waste aspiration steps used syringe motor speeds set at 25% of normal to minimize foaming of the kit reagent and to eliminate aspiration of the paper disc. (3) MPII Automated Heater Controller and PCR96 Heat Transfer Block. This softwarecontrolled device can heat up to four heater tiles independently and is used for drying washed FTA punches (Fig. 2). The off-deck controller preserves deck space. The heater tile with a PCR plate and PCR96 heat transfer block assembly is placed on the main deck. (4) 175 mL RoboRack Filter Disposable Tips. Barrier tips prevent cross-contamination when processing multiple samples. Fig. 3 shows the deck layout as it appears in the WinPREP software test outline. Note the location of the heater tile assembly. The workstation also included the Gripper Integration Platform and DPC MicroMix 5 Shaker
AUTOMATED FORENSIC DNA PURIFICATION
185
Fig. 1. Automated FTA DNA purification procedure.
(Diagnostics Products Corporation, Los Angeles, California) Options. Neither is required for a one-plate protocol. Optimized FTA protocol for identifiler analysis Four reference bloodstained FTA card samples of known genotypes were obtained from the FBI and processed manually in 1.5-mL-conical tubes. To optimize for analysis with the Identifiler Kit [5,6], three different DNA purification protocols and various STR PCR setup methods were compared.
186
TACK
et al
Fig. 2. Automated MultiPROBE II Heater Controller with quad heater tiles.
Variables tested are shown below. All samples were analyzed on either an ABI PRISM 310 or 3100 Genetic Analyzer using GeneScan and Genotyper software. The following methods were tested: (1) The Whatman 2003 FTA application note method, (2) the ABI-modified FTA method (ABI customer support), and (3) the NaOH (10 mM) wash method [2]. The variables tested were punch sizes (1.2 mm, 2 mm); wash conditions (FTA buffer, 10 mM NaOH, TE-1, water); drying (air-dry or 50 C heater);
Fig. 3. MultiPROBE II FTA DNA purification protocol deck layout.
AUTOMATED FORENSIC DNA PURIFICATION
187
Fig. 4. Genotyper electropherogram for reference sample 4.
STR kits (Identifiler (15 STR loci þ amelogenin), Profiler Plus (9 STR loci þ amelogenin) PCR reaction volumes (15 mL (undiluted), 25 mL (þ 10 mL TE-1) and PCR cycles (28 or 24). The ABI-modified FTA method 2, when used with Identifiler amplification, gave the best results. The overall processing scheme used with method 2 is as follows: Prepare 1.2 mm FTA punches using a Harris Micro Punch in PCR microfuge tubes. Wash twice with 180 mL FTA Purification Reagent: vortex, incubate 5 minutes, aspirate supernatant. Wash once with 180 mL TE-1: vortex, incubate 5 minutes, aspirate supernatant. Wash once with H2O: vortex, incubate 5 minutes, aspirate supernatant. Dry at 50 C for 10–15 minutes. Add 15 mL Identifiler master mix (no water added to sample disc). Amplify 24 cycles using kit recommended cycling parameters in an Applied Biosystems GeneAmp PCR System 2700 (Applera Corporation, Foster City, California). Add size standards. Denature and load 1 mL on CE system. All samples processed showed sharp well-resolved peaks with high sensitivity (w 3000–8000 RU). All 15 STR alleles were easily called using Genotyper software. Optimum conditions included partial volume PCR
188
TACK
et al
Fig. 5. (A) Checkerboard plate format used for cross-contamination analysis; (B) Cross-contamination assay results. Yes ¼ correct STR profile; bl ¼ blank profile with no STR peaks but standards are present.
conditions (15 mL master mix, undiluted) with PCR cycle number n ¼ 24. Note: samples processed using NaOH wash method 3 showed well-resolved peaks with all 15 STR alleles called correctly, but with much reduced peak heights compared to method 2 (not shown). Automated FTA DNA purification protocol Reference sample punches were processed using the MultiPROBE II workstation and a WinPREP template created using method 2 parameters (see Fig. 1). Discs were placed in a 96-well PCR plate on the heater tile assembly located on the main deck (see Fig. 3). After washing, the sample plate was automatically heated to 50 C. All waste removal steps used 25% of normal aspirate motor speeds to minimize disk contact and avoid aspirating punches during the wash steps. Reagents were replicate-dispensed using fixed tip mode at 101% well height to maximize throughput without contamination. Reagent waste was removed using disposable filter tips. The processing time is w 30 minutes for 96 samples, not including a 20 minutes
AUTOMATED FORENSIC DNA PURIFICATION
189
automated drying step. About 20 minutes was dedicated to soak time for the several washes used. Purified DNA is ready for immediate STR analysis. Identifiler master mix was added to each well and samples were amplified and analyzed on an ABI Prism 3100 Genetic Analyzer. A typical electropherogram for a purified punch sample is shown in Fig. 4 and displays the 15 STR loci and the Amelogenin locus expected. Each dye panel is shown separately; the orange LIZ size standard peaks are shown in the bottom panel. Potential cross-contamination of samples was evaluated using a checkerboard pattern of reference punches and empty wells in a PCR plate as shown in Fig. 5A. Pink wells each contained a different known bloodstained punch. Remaining wells are empty blanks. After processing using the MultiPROBE II workstation, both sample and blank wells were analyzed using Identifiler analysis. The STR analysis of FTA reference sample 2 processed in well C3 is shown in Fig. 6A. STR analysis results for blank well B3 are
Fig. 6. (A) FTA sample STR analysis results for well C3; (B) blank sample STR analysis results for well B3.
190
TACK
et al
shown in Fig. 6B. B3 is adjacent to well C3. The 5-dye set for B3 is displayed as a single panel. Only the LIZ dye standard peaks added to each sample before CE analysis are present, as expected. The results for one experiment are shown in Fig. 5B. No evidence of cross-contamination was found in any sample analyzed. The reference samples all gave the correct STR profiles. No STR peaks were observed in any blank wells. Peak height, stutter percentages, and overall quality of electropherograms using Identifiler and Profiler Plus [7] kits were identical or better than manual sample purification. Fig. 7 shows a summary of the Identifiler alleles, correctly called for four bloodstained FTA reference samples purified using the MultiPROBE II Forensic Workstation and reagents from the Whatman FTA Purification Kit.
Summary We describe a rapid, simple, and reliable procedure for automated processing of 1.2 mm punches from FTA paper stains for PCR-based STR analysis. The robotic procedure uses Whatman FTA Purification Kit reagents and was optimized for use with Identifiler STR typing. Comparable results were obtained with Profiler Plus kits. All DNA STR profiles were compared against results obtained using traditional extraction techniques and were congruent at all loci tested. The protocol is easily scalable. The 30 min/plate run time is decreased if multiple plates are processed at once. Punches 1.2 mm in size provide ample DNA for multiple STR analysis runs. The larger 2 mm bloodstained punches did not give as good results using the Identifiler kit. Fully automated drying using software-directed temperature control is available with the heater option. The Gripper Integration Platform option is not required for processing small plate numbers but may
Fig. 7. Reference sample STR typing results.
AUTOMATED FORENSIC DNA PURIFICATION
191
be used to move plates to the off-deck heater or for repopulating the deck if processing large plate batches. The same robot system can be used to automate other forensic sample protocols. This includes DNA quantification, normalization, dilution, PCR setup assays, and the Promega DNA IQ System Kit for casework sample DNA isolation.
Acknowledgments We gratefully thank the following: P. W. Boonlayangoor, Ph.D., Laboratory Director, Independent Forensics of Illinois, Lombard, IL; Rob McPheeters, Automations Specialist, Genetic ID, Whatman, Inc., Clifton, NJ; Xavier Aranda, DNA Identity Lab, University of North Texas, Fort Worth, TX; and ABI Technical Support at Applied Biosystems Inc., Foster City, CA.
References [1] Whatman Inc. FTA Protocols: Collect, Transport, Archive and Access Nucleic Acids - All at Room Temperature; WB120047; Copyright Whatman Inc., 2002. Available at: www. whatman.com. [2] Smith LM, Burgoyne LA. Collecting, archiving and processing DNA from wildlife samples using FTA databasing paper. BMC Ecology 2004;4:4–15. [3] Whatman Inc. Preparation of Isolated DNA from FTA Cards Using an Automated Liquid Handling System; Application Note 51609; Copyright Whatman Inc., 2003. [4] Smith MA, Igoe F, Pierce K, et al. FTA Technology – New Advances for the Collection, Shipment, Archiving and Processing of Biological Samples for Human Identification. Presented at the 13th International Symposium on Human Identification, Phoenix AZ, 2002; Poster Abstract. [5] Applied Biosystems. AmpFlSTR Identifiler PCR Amplification Kit; Human Identification Publication 112PB02-03; Copyright Applied Biosystems 2002. [6] Applied Biosystems. AmpFlSTR Identifiler PCR Amplification Kit User’s Manual; Product Manual PPN 4323291; Copyright Applied Biosystems 2002. [7] Applied Biosystems. AmpFlSTR Profiler PCR Amplification Kit; Human Identification Publication 112PB02-01; Copyright Applied Biosystems 2002.