Accepted Manuscript Title: Direct amplification of casework bloodstains using the Promega PowerPlex® 21 PCR Amplification System Author: Kerryn Gray Damian Crowle Pam Scott PII: DOI: Reference:
S1872-4973(14)00095-7 http://dx.doi.org/doi:10.1016/j.fsigen.2014.05.003 FSIGEN 1154
To appear in:
Forensic Science International: Genetics
Received date: Revised date: Accepted date:
20-10-2013 23-3-2014 5-5-2014
Please cite this article as: K. Gray, D. Crowle, P. Scott, Direct amplification of casework bloodstains using the Promega PowerPlexregd 21 PCR Amplification System, Forensic Science International: Genetics (2014), http://dx.doi.org/10.1016/j.fsigen.2014.05.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Direct amplification of casework bloodstains using the Promega PowerPlex® 21 PCR Amplification System Kerryn Gray*, Damian Crowle, Pam Scott
Forensic Science Service Tasmania, 20 St Johns Avenue, New Town, Tasmania 7008, Australia
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*Corresponding author: Tel.: +61 3 62 785 677. E-mail address: [email protected] (K. Gray).
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Direct amplification of casework bloodstains using the Promega PowerPlex® 21 PCR Amplification System
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1. Introduction
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Keywords: STR Direct amplification Casework bloodstains PowerPlex 21
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ABSTRACT A significant number of evidence items submitted to Forensic Science Service Tasmania (FSST) are blood swabs or bloodstained items. Samples from these items routinely undergo phenol:chloroform:isoamyl alcohol organic extraction and quantitative Polymerase Chain Reaction (qPCR) testing prior to PowerPlex® 21 amplification. This multistep process has significant cost and timeframe implications in a fiscal climate of tightening government budgets, pressure towards improved operating efficiencies, and an increasing emphasis on rapid techniques better supporting intelligence-led policing. Direct amplification of blood and buccal cells on cloth and Whatman FTA™ card with PowerPlex® 21 has already been successfully implemented for reference samples, eliminating the requirement for sample pre-treatment. Scope for expanding this method to include less pristine casework blood swabs and samples from bloodstained items was explored in an endeavor to eliminate lengthy DNA extraction, purification and qPCR steps for a wider subset of samples. Blood was deposited onto a range of substrates including those historically found to inhibit STR amplification. Samples were collected with micro-punch, micro-swab, or both. The potential for further fiscal savings via reduced volume amplifications was assessed by amplifying all samples at full and reduced volume (25 and 13µL). Overall success rate data showed 80% of samples yielded a complete profile at reduced volume, compared to 78% at full volume. Particularly high success rates were observed for the blood on fabric/textile category with 100% of micro-punch samples yielding complete profiles at reduced volume and 85% at full volume. Following the success of this trial, direct amplification of suitable casework blood samples has been implemented at reduced volume. Significant benefits have been experienced, most noticeably where results from crucial items have been provided to police investigators prior to interview of suspects, and a coronial identification has been successfully completed in a short timeframe to avoid delay in the release of human remains to family members.
In recent times, the forensic community and law enforcement agencies have been placing increased emphasis on the development of rapid, compact and portable devices capable of producing real-time results from forensic samples at the crime scene [1-4]. The potential for DNA profiling to shift from being a piece of the puzzle investigators may wait weeks or months for, to being an intelligence tool used in the crucial early stages of an investigation is an attractive prospect [5]. Although the potential for these technologies to revolutionise DNA testing in field applications is great, in such settings there remains a number of challenges around profile interpretation, affordability, validation and quality assurance and as such there has been a lack of widespread adoption by law enforcement agencies to date [6]. Rapid laboratory based methods have the benefit of a controlled facility supported by well founded quality assurance systems. Rapid multiplex PCR amplification and direct amplification are two such methods, also forming the basis of some field units [4,7]. Standard PCR run times have traditionally been upwards of 3 hours; for one multiplex kit this has successfully been reduced to less than 36 minutes [8,9]. Direct amplification is becoming increasingly popular for reference blood and buccal cell on FTA card samples and in recent times has been successfully applied to a single hair follicle and tape lifts of clothing and swabs [10-17]. If standard kits are used for direct and rapid amplification, resultant profiles will be comparable with searchable databases of profiles from routine casework. This is a notable advantage over many field-based methods containing significantly reduced marker numbers, capability only to screen for the presence of genetic material, or significant limitations to simultaneous sample processing capability [1,3,7,18]. With improving technology and the availability of a new generation of robust, inhibitor tolerant STR kits, the ®
potential exists for direct amplification of a larger subset of casework samples. The Promega PowerPlex 21 PCR Amplification System (PP21) is actively marketed for its increased inhibitor tolerance, sub 90 minute rapid PCR protocol and compatibility with direct amplification of Whatman FTA™ (FTA) card punches and pretreated swabs [19]. These features enable DNA profiling results from reference samples to be available within a matter of hours from start
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to finish. Scope for expanding the eligible sample types to include casework bloodstains and blood swabs was investigated despite the manufacturer suggesting these types of samples require pretreatment with SwabSolution™ prior to amplification [20]. An attractive potential exists for the rapid processing of samples recovered from serious assault and homicide scenes where significant bloodshed has occurred. Time pressure can be considerable under these circumstances and rapid screening of bloodstains may be crucial to the momentum of the investigation. PP21 has already proven its robust ability to amplify casework sample types that are problematic to purify and failed to yield profiles using other STR kits. On a number of occasions PP21 has successfully amplified poor quality samples that completely inhibited the Promega Plexor® HY qPCR reaction. Its ability to consistently obtain complete profiles from direct amplification of unpurified blood on cloth and FTA card is also noteworthy, in that the heme compound in blood has long been regarded as a major inhibitor of PCR and usually requires removal prior to achieving effective amplification [21]. Other commonly encountered PCR inhibitors include wood, leather, soil, sand, leaf litter and textile dyes [22,23]. The ubiquitous existence of these inhibitors in the environment and the high incidence of their co-recovery with target biological material in forensic samples is a significant drawback of traditional STR typing methods [23,24]. Reduced volume amplifications present an additional avenue for consideration [25]. PP21 has demonstrated a flexible range of working amplification volumes in previous internal validation studies, with successful reduction to 13µL for reference samples. 2. Materials and methods
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Forty five mock crime scene samples were prepared by spotting an unmeasured amount of fresh whole human blood (staff donors) onto a range of substrates (Fig. 1 and Table 1) including those historically known to inhibit STR amplification at FSST, such as denim, plant materials, wood, metal and rocks. A diverse range of substrates of varying porosity and quality were selected to challenge the robustness of the new method and assess any limitations. Samples were left to dry for two hours. Stains were sampled with a micro-swab (Copan, part no. 8164CIS) moistened with sterile water; or 1.0mm and 1.2mm Harris Micro-Punches (Whatman, part nos. WHAWB100060 and WHAWB100061); or a combination of all three sampling methods for success rate comparison. Samples were placed into a MicroAmp® Optical 96-well Reaction Plate (Applied Biosystems®, part no. N8010560) and amplified with the PowerPlex® 21 PCR amplification system at reaction volumes of 25 and 13µL for micro-swabs, 25µL for 1.2mm micropunches and 13µL for 1.0mm micro-punches. Differing punch sizes were used for each reaction volume based on previous validation work for the direct amplification of reference samples. For micro-swabs, a very small (1-2mm) piece of swab material was excised from the stained region of the swab tip and was placed into the amplification reaction mixture. Micro-swab samples for reduced volume amplification were sampled marginally smaller than for full volume amplification, with optimum sample size estimated based on the colour and intensity of the bloodstain, and amplification volume. Actual amounts of DNA added to the amplification reaction were not quantified. Amplification was performed using Veriti® thermal cyclers following the manufacturer’s recommended direct amplification protocol internally validated to 27 cycles [20]. Amplified samples were prepared for capillary electrophoresis following the manufacturer’s recommended guidelines and injected on an Applied Biosystems® 3130xl Genetic Analyser at 3kV for 5 and 1 seconds [20]. Analysis and genotyping of data was performed using GeneMapper® ID v3.2.1 software with panels and bins supplied by Promega. Samples were genotyped using internally validated 20 RFU analysis and 100 RFU homozygote thresholds. Overall success rates were determined for full and reduced volume amplifications by calculating the percentage of complete profiles obtained using each method. Success rates were also calculated for the alternate sampling methods (micro-swab versus micro-punch). 3. Results and discussion
Overall success rates determined for both amplification volumes showed little difference, with complete profiles obtained from 80% of samples amplified at reduced volume, compared to 78% at full volume (Table 1). Partial profiles were also very informative, returning a high number of reportable alleles (≥31 alleles from a possible 42 per profile) with the exception of the reduced volume blood on leather micro-punch sample (23 alleles). For this and other blood on non-porous substrate samples (leaf, leather, rubber and latex gloves), more complete profiles were obtained from micro-swabs of the stains rather than micro-punches. Some difficulty was encountered sampling these substrates by micro-punch as blood tended to flake off once dried, making it problematic to capture the desired amount and successfully transfer it to the amplification mixture. The average peak height ratio (PHR) was determined from the heterozygous loci for full and reduced volume amplifications with the variable amounts of input DNA. On average, PHR ranged from 0.81 (D2S1338 and D6S1043) to 0.91 (TH01, vWA and D8S1179) for full volume reactions and 0.79 (D2S1338) to 0.92 (D8S1179) for reduced volume reactions. Other rapid amplification methods have reported PHRs on average above 0.85 [9], and ranging from 0.84
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to 0.92 [8] with ideal amounts of input DNA (≥0.5ng). Inter-locus balance was calculated as the total RFU value of a locus divided by the total RFU value of the profile (for a 21 locus profile each locus would ideally show 0.048 of the total RFU value). Averages ranged from 0.015 (TPOX) to 0.085 (vWA) for full volume reactions and 0.014 (TPOX) to 0.096 (D12S391) for reduced volume reactions. These ranges are also similar to those reported for other rapid methods [16]. Different sized micro-punches (1.2mm for 25µL amplifications and 1mm for 13µL amplifications) were used based on previous in-house optimisation studies for direct amplification of FTA cards which showed a reduction in amplification volume requires a reduction in DNA input amount. Approximately equivalent peak heights are obtained from a 1.0mm micro-punch amplified at 13µL and a 1.2mm micro-punch amplified at 25µL (data not shown). A degree of variation existed in the size of the piece of stained micro-swab material excised for amplification as a result of varying substrate quality and dilution of blood with impurities such as dirt and rust. Typical bloodstain colouration was difficult to distinguish on some of these dirtier swabs; not dissimilar to many casework samples. As a consequence, samples closer to 2mm were taken from these swabs, resulting in noticeably discoloured and coagulated PCR product in some instances (Fig. 2 and Fig. 3C). Results showed this had negligible impact on the ability of PP21 to yield a profile, with the kit demonstrating a robust nature in the presence of noticeable impurities and variable amounts of input DNA. Mock crime scene samples were left to dry for 2 hours, a disparity to actual casework samples where collection would generally occur some hours or days after being deposited, and sometimes much longer. Retrospectively, a longer drying time could have been allowed for these samples to more accurately mimic real casework. Subsequent analysis of casework samples (described following) has shown no disparity between success rates for trial samples and casework samples. As such, it appears, drying time has no effect on success rates. 3.1 Casework application
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Since validation and implementation of this method for amplification of casework blood samples at FSST, 340 samples have been processed. Samples must meet specific requirements to be eligible for direct amplification, namely the stain must be of sufficient size to take additional samples for organic extraction or third party retesting if required, and the presence of blood must be confirmed. For major crime samples the presence of human blood is confirmed using the ABAcard® Hematrace® test kit (Abacus Diagnostics®, part no. 708424) and for minor crime samples the haemochromogen confirmatory test for blood is used [26]. Some improvements to downstream processing have been made to minimise contamination opportunities. Disposable 1mm medical biopsy punches with plunger (Miltex, part no. 33-31AA-P) have been introduced for individual sampling of blood on porous substrates. All samples are placed into individually capped 0.2mL MicroAmp® tubes (Applied Biosystems®, part no. N8010612), stored at -80°C (if required), then carried through the amplification process without the need for sample transfer to a 96-well reaction plate. Blood swabs are sampled as previously described, by cutting a small (1-2mm) piece of the stain and placing it into a labelled 0.2mL MicroAmp® tube (Fig. 3). All samples are amplified with PP21 at reduced volume (13µL), by addition of amplification mix directly into the sample tube and amplification and capillary electrophoresis performed as earlier described. Use of 5 and 1 second injection times enables a greater number of complete profiles to be captured and is necessary given the variable and uncontrollable amounts of DNA being amplified. Introduction of direct amplification and the associated alternative sampling methods has resulted in additional onus on examiners to make an assessment of the suitability of a stain for direct amplification and sample accordingly. After an initial familiarisation period, examiners quickly became comfortable with the sampling method, success rates, the generally variable nature of the stains and optimum sample size, with no issues reported by examiners using the method. Of the 340 samples tested thus far, complete single person profiles were obtained from 70% of samples, partial single person profiles from 8%, mixed profiles from 16%, predominantly dye-blob artefacts from 1%, 1% were overloaded with DNA, and 4% gave no reportable alleles (data not shown). Overall success rates showed 90% of samples yielded an acceptable profile in the first instance and 10% required subsequent organic extraction and qPCR. Acceptable results included some mixed and partial profiles, as partial profiles are generally adequate for matching or exclusionary purposes. During the initial stages of implementation, genotyping was restricted to apparent single person profiles; however standard laboratory mixture interpretation guidelines have now been validated to include genotyping of mixed profiles generated from direct amplification at 27 cycles. Of the 10% (33 samples) to undergo subsequent organic retesting, 31 produced a greater number of reportable alleles. A notable observation has been the negligible effect of stain intensity. Many swabs exhibited pale or dilute bloodstain colouration and complete profiles were still obtained. Similarly good results were obtained from light transfer bloodstains on the surface of clothing where the blood had not penetrated the weave of the fabric to any significant degree. Somewhat surprisingly, the variable nature of casework samples compared to reference samples appears to be of very little consequence.
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Direct amplification is not routinely utilised in-house for its reduced processing time. Samples suitable for direction amplification are stored temporarily and amplified once a small batch of up to 15-30 samples has been filled. Results are then absorbed into standard reporting processes with standard turn around times; reduced processing cost being the main benefit. The true value however, undoubtedly lies where there is an investigative urgency for results from critical samples. Samples are run regardless of batch size and results are provided to police immediately following routine quality assurance checks. For a recent murder case, intelligence level results were provided to detectives in the early stages of the investigation, prior to the interview of suspects. For a recent coronial matter, a priority request was received from the coroners’ office for DNA identification of decomposing human remains, where fingerprint and dental identification were not possible. Due to the level of decomposition, organic extraction of blood from the deceased was attempted in the first instance and yielded no reportable alleles. Given standard extraction timeframes, repeat organic extraction would not normally commence until the following week. Direct amplification was therefore attempted on two more blood samples. Results were obtained by 12pm, with both samples again failing. A sample of spleen from the deceased was then prepared for direct amplification and processed during the afternoon, producing a partial 38 allele profile with a match probability of less than 1 in 100 billion (FSST default). Match details were provided to the coroners’ office on the Friday afternoon, enabling the remains of the deceased to be released to family members without unnecessary delay. The method has also been particularly beneficial for a number of serious assault cases where screening of the victims clothing, heavily stained with the victims own blood, was necessary to identify the presence of blood from the offender. In the above instances, many samples could be taken from bloodstained items and rapidly processed by direct amplification. Processing of suitable casework bloodstains using reduced volume direct amplifications has resulted in nearly a two and a half fold decrease in processing cost per sample. This represents the cost of reagents and consumables and is inclusive of the cost of retesting 10% of samples via the standard organic and qPCR method. The cost of a scientists’ time has not been included, as quantifying these additional savings is difficult to do accurately, although anecdotal evidence suggests a saving of 3 to 4 hours of hands on time. The benefits presented thus far apply broadly in the forensic context regardless of extraction and qPCR chemistries being utilised. Most forensic facilities employ alternative extraction chemistries on automated platforms. These processes are equally as time consuming and costly, and could be bypassed entirely for suitable bloodstains. Increased total laboratory throughput capability is also enabled through the availability of standard extraction places previously occupied by these samples.
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The Promega PowerPlex® 21 PCR Amplification System has demonstrated a clear ability to handle overtly impure casework blood samples and produce complete profiles from direct amplification in less than 3 hours. This has significantly reduced the time and expense required to routinely process samples by removing the requirement for extraction and qPCR. There are obvious benefits to laboratory operations in implementing direct amplification of these sample types, with reduced volume reactions also providing a clear avenue for further fiscal savings. In a non fiscal context, direct amplification has provided significant support for intelligence-led policing and will undoubtedly continue be an advantageous tool in the critical early stages of criminal investigations. Acknowledgements
The authors would like to thank Anna Lemalu for experimental assistance; staff blood donors; and Dr Jason Buchan, Dr Louise McMahon and the technical reviewers for greatly improving this paper through peer review. References
[1] P. Liu, T.S. Seo, N. Beyor, K-J. Shin, J.R. Scherer, R.A. Mathies, Integrated Portable Polymerase Chain ReactionCapillary Electrophoresis Microsystem for Rapid Forensic Short Tandem Repeat Typing, Anal. Chem. 79 (5) (2007) 1881-1889. [2] C.A. Batt, S.J. Stelick, M.J. Kennedy, C.S. Lui, A.J. Lowe, A Hand-held DNA Based Forensic Tool, Research Report, (internet) 2009, (cited: March 5, 2014). Available from: https://www.ncjrs.gov/pdffiles1/nij/grants/227499.pdf [3] N. Dawnay, B. Stafford-Allen, D. Moore, S. Blackman, P. Rendell, E.K. Hanson, J. Ballantyne, B. Kallifatidis, J. Mendel, D.K. Mills, R. Nagy, S. Wells, Developmental Validation of the ParaDNA® Screening System – A
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[10] R. Weispfenning, K. Oostdik, M. Ensenberger, B. Krenke, C. Sprecher, D. Storts, Doing more with less: Implementing direct amplification with the PowerPlex® 18D System, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) e409-e410.
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[11] M. Stene, A. Buchard, C. Børsting, N. Morling, Validation of the AmpFlSTR® Identifiler® Direct PCR Amplification kit in a laboratory accredited according to the ISO17025 standard, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) e165e166.
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[12] D.Y. Wang, C-W. Chang, N.J. Oldroyd, L.K. Hennessy, Direct amplification of STRs from blood or buccal cell samples, Forensic Sci. Int. Genet. Suppl. Ser. 2 (2009) 113-114. [13] P.M. Vallone, C.R. Hill, E.L.R. Butts, Concordance study of direct PCR kits: PowerPlex 18D and Identifiler Direct, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) e353-e354. [14] B.A. Myers, J.L. King, B. Budowle, Evaluation and comparative analysis of direct amplification of STRs using PowerPlex® 18D and Identifiler® Direct systems, Forensic Sci. Int. Genet. (2012) 640-645. [15] R. Ottens, D. Taylor, D. Abarno, A. Linacre, Successful direct amplification of nuclear markers from a single hair follicle, Forensic Sci. Med. Pathol. 9 (2) (2013) 238-243. [16] S. Verheij, J. Harteveld, T. Sijen, A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples, Forensic Sci. Int. Genet. (2012) 167-175. [17] A. Linacre, V. Pekarek, Y.C. Swaran, S.S. Tobe, Generation of DNA profiles from fabrics without DNA extraction, Forensic Sci. Int. Genet. (2010) 137-141. [18] RapidHIT™ Developmental Validation and Applications Summary, (internet) 2013, (cited: March 7, 2014). Available from: http://integenx.com/wp-content/uploads/2013/09/Developmental-Validation-Data-Summary.pdf [19] M.G. Ensenberger, P.M. Fulmer, B.E. Krenke, R.S. McLaren, C.J. Spreacher, D.R. Storts, Development of the PowerPlex® 21 System, Profiles in DNA (2012) (internet) (cited: September 4, 2013). Available from: http://au.promega.com/resources/articles/profiles-in-dna/2012/development-of-the-powerplex-21-system/
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[20] PowerPlex® 21 System Technical Manual, Revision 7/12, (internet) 2012, (cited: September 5, 2013). Available from: http://www.promega.com/~/media/Files/Resources/Protocols/Technical%20Manuals/101/PowerPlex%2021%20 System%20Protocol.pdf [21] A. Akane, K. Matsubara, H. Nakamura, S. Takahashi, K. Kimura, Identification of the Heme Compound Copurified with Deoxyribonucleic Acid (DNA) from Bloodstains, a Major Inhibitor of Polymerase Chain Reaction (PCR) Amplification, J. Forensic Sci. 39 (2) (1994) 362-372.
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[26] A.L. Hatch, A modified Reagent for the Confirmation of Blood, J. Forensic Sci 38 (6) (1993) 1502-1506.
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Table 1 Allele count comparison for all sampling and amplification methods (partial profiles highlighted). A complete PP21 profile has 42 alleles.
Toilet paper Paper towel Tissue Plain white paper Cardboard Satin fabric with interfacing Polyester/elastane fabric A Polyester/elastane fabric B Polyester/elastane fabric (bra) Natural cotton fabric Drill fabric Flannelette fabric Denim fabric Shantung fabric Thermal knit fabric Cotton twill Towelling Woollen beanie Woollen sock Polyester/cotton fabric Chiffon fabric Satin fabric Textile batting Carpet fibres ‘Chux’ cloth Grass Leaf Leather Rubber glove Latex examination glove Press seal plastic bag Plastic supermarket bag Metal spatula Glass Metal bracket Painted wood Copper Bullet Rock Concrete Rusty drain grate Asphalt Garden bark Treated pine Paving stone
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Miscellaneous - non-porous (micro-punch & micro-swab or micro-swab only)
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Fabric/textile - porous (micro-punch only)
Hard - porous (micro-swab only)
% complete profiles
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Paper based - porous (micro-punch only)
25µL amplification 1.2mm Cut piece punch of swab 42 42 37 42 40 42 42 42 42 42 42 42 42 42 42 42 42 42 42 41 41 37 42 42 42 42 42 35 42 41 42 37 42 42 42 42 42 42 42 42 42 41 33 42 42 42 42 42 42 41 73 85 78
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Substrate description
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Substrate type
13µL amplification 1.0mm Cut piece punch of swab 42 42 42 37 33 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 31 42 23 42 37 41 37 42 42 42 42 42 42 42 41 32 42 42 42 42 42 42 41 80 80 80
- Indicates sampling method not tested.
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We report on the successful direct amplification of casework blood samples. DNA extraction and quantitation are not required regardless of sample purity. Amplification volume has successfully been reduced. Rapid turn-around times have been achieved in support of intelligence-led policing. Close to a two and a half fold decrease in processing cost has been achieved.
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Additional Files
Fig. 1. Subset of samples tested Fig. 2. 96-well PCR product plate illustrating variable sample colour and degrees of impurity. Numbers indicate allele counts (42 = full profile; control sample allele counts omitted)
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Fig. 3. Mock casework example of direct amplification. (A) dried bloodstain on asphalt (B) micro-swab of bloodstain on asphalt with 1-2mm portion of stain excised from swab head (C) 0.2mL MicroAmp PCR product tube containing excised sample and 13µL of amplification mix (D) Resultant electropherogram (complete profile).
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S1872-4973(14)00095-7 http://dx.doi.org/doi:10.1016/j.fsigen.2014.05.003 FSIGEN 1154
To appear in:
Forensic Science International: Genetics
Received date: Revised date: Accepted date:
20-10-2013 23-3-2014 5-5-2014
Please cite this article as: K. Gray, D. Crowle, P. Scott, Direct amplification of casework bloodstains using the Promega PowerPlexregd 21 PCR Amplification System, Forensic Science International: Genetics (2014), http://dx.doi.org/10.1016/j.fsigen.2014.05.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Direct amplification of casework bloodstains using the Promega PowerPlex® 21 PCR Amplification System Kerryn Gray*, Damian Crowle, Pam Scott
Forensic Science Service Tasmania, 20 St Johns Avenue, New Town, Tasmania 7008, Australia
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*Corresponding author: Tel.: +61 3 62 785 677. E-mail address: [email protected] (K. Gray).
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Direct amplification of casework bloodstains using the Promega PowerPlex® 21 PCR Amplification System
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1. Introduction
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Keywords: STR Direct amplification Casework bloodstains PowerPlex 21
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ABSTRACT A significant number of evidence items submitted to Forensic Science Service Tasmania (FSST) are blood swabs or bloodstained items. Samples from these items routinely undergo phenol:chloroform:isoamyl alcohol organic extraction and quantitative Polymerase Chain Reaction (qPCR) testing prior to PowerPlex® 21 amplification. This multistep process has significant cost and timeframe implications in a fiscal climate of tightening government budgets, pressure towards improved operating efficiencies, and an increasing emphasis on rapid techniques better supporting intelligence-led policing. Direct amplification of blood and buccal cells on cloth and Whatman FTA™ card with PowerPlex® 21 has already been successfully implemented for reference samples, eliminating the requirement for sample pre-treatment. Scope for expanding this method to include less pristine casework blood swabs and samples from bloodstained items was explored in an endeavor to eliminate lengthy DNA extraction, purification and qPCR steps for a wider subset of samples. Blood was deposited onto a range of substrates including those historically found to inhibit STR amplification. Samples were collected with micro-punch, micro-swab, or both. The potential for further fiscal savings via reduced volume amplifications was assessed by amplifying all samples at full and reduced volume (25 and 13µL). Overall success rate data showed 80% of samples yielded a complete profile at reduced volume, compared to 78% at full volume. Particularly high success rates were observed for the blood on fabric/textile category with 100% of micro-punch samples yielding complete profiles at reduced volume and 85% at full volume. Following the success of this trial, direct amplification of suitable casework blood samples has been implemented at reduced volume. Significant benefits have been experienced, most noticeably where results from crucial items have been provided to police investigators prior to interview of suspects, and a coronial identification has been successfully completed in a short timeframe to avoid delay in the release of human remains to family members.
In recent times, the forensic community and law enforcement agencies have been placing increased emphasis on the development of rapid, compact and portable devices capable of producing real-time results from forensic samples at the crime scene [1-4]. The potential for DNA profiling to shift from being a piece of the puzzle investigators may wait weeks or months for, to being an intelligence tool used in the crucial early stages of an investigation is an attractive prospect [5]. Although the potential for these technologies to revolutionise DNA testing in field applications is great, in such settings there remains a number of challenges around profile interpretation, affordability, validation and quality assurance and as such there has been a lack of widespread adoption by law enforcement agencies to date [6]. Rapid laboratory based methods have the benefit of a controlled facility supported by well founded quality assurance systems. Rapid multiplex PCR amplification and direct amplification are two such methods, also forming the basis of some field units [4,7]. Standard PCR run times have traditionally been upwards of 3 hours; for one multiplex kit this has successfully been reduced to less than 36 minutes [8,9]. Direct amplification is becoming increasingly popular for reference blood and buccal cell on FTA card samples and in recent times has been successfully applied to a single hair follicle and tape lifts of clothing and swabs [10-17]. If standard kits are used for direct and rapid amplification, resultant profiles will be comparable with searchable databases of profiles from routine casework. This is a notable advantage over many field-based methods containing significantly reduced marker numbers, capability only to screen for the presence of genetic material, or significant limitations to simultaneous sample processing capability [1,3,7,18]. With improving technology and the availability of a new generation of robust, inhibitor tolerant STR kits, the ®
potential exists for direct amplification of a larger subset of casework samples. The Promega PowerPlex 21 PCR Amplification System (PP21) is actively marketed for its increased inhibitor tolerance, sub 90 minute rapid PCR protocol and compatibility with direct amplification of Whatman FTA™ (FTA) card punches and pretreated swabs [19]. These features enable DNA profiling results from reference samples to be available within a matter of hours from start
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to finish. Scope for expanding the eligible sample types to include casework bloodstains and blood swabs was investigated despite the manufacturer suggesting these types of samples require pretreatment with SwabSolution™ prior to amplification [20]. An attractive potential exists for the rapid processing of samples recovered from serious assault and homicide scenes where significant bloodshed has occurred. Time pressure can be considerable under these circumstances and rapid screening of bloodstains may be crucial to the momentum of the investigation. PP21 has already proven its robust ability to amplify casework sample types that are problematic to purify and failed to yield profiles using other STR kits. On a number of occasions PP21 has successfully amplified poor quality samples that completely inhibited the Promega Plexor® HY qPCR reaction. Its ability to consistently obtain complete profiles from direct amplification of unpurified blood on cloth and FTA card is also noteworthy, in that the heme compound in blood has long been regarded as a major inhibitor of PCR and usually requires removal prior to achieving effective amplification [21]. Other commonly encountered PCR inhibitors include wood, leather, soil, sand, leaf litter and textile dyes [22,23]. The ubiquitous existence of these inhibitors in the environment and the high incidence of their co-recovery with target biological material in forensic samples is a significant drawback of traditional STR typing methods [23,24]. Reduced volume amplifications present an additional avenue for consideration [25]. PP21 has demonstrated a flexible range of working amplification volumes in previous internal validation studies, with successful reduction to 13µL for reference samples. 2. Materials and methods
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Forty five mock crime scene samples were prepared by spotting an unmeasured amount of fresh whole human blood (staff donors) onto a range of substrates (Fig. 1 and Table 1) including those historically known to inhibit STR amplification at FSST, such as denim, plant materials, wood, metal and rocks. A diverse range of substrates of varying porosity and quality were selected to challenge the robustness of the new method and assess any limitations. Samples were left to dry for two hours. Stains were sampled with a micro-swab (Copan, part no. 8164CIS) moistened with sterile water; or 1.0mm and 1.2mm Harris Micro-Punches (Whatman, part nos. WHAWB100060 and WHAWB100061); or a combination of all three sampling methods for success rate comparison. Samples were placed into a MicroAmp® Optical 96-well Reaction Plate (Applied Biosystems®, part no. N8010560) and amplified with the PowerPlex® 21 PCR amplification system at reaction volumes of 25 and 13µL for micro-swabs, 25µL for 1.2mm micropunches and 13µL for 1.0mm micro-punches. Differing punch sizes were used for each reaction volume based on previous validation work for the direct amplification of reference samples. For micro-swabs, a very small (1-2mm) piece of swab material was excised from the stained region of the swab tip and was placed into the amplification reaction mixture. Micro-swab samples for reduced volume amplification were sampled marginally smaller than for full volume amplification, with optimum sample size estimated based on the colour and intensity of the bloodstain, and amplification volume. Actual amounts of DNA added to the amplification reaction were not quantified. Amplification was performed using Veriti® thermal cyclers following the manufacturer’s recommended direct amplification protocol internally validated to 27 cycles [20]. Amplified samples were prepared for capillary electrophoresis following the manufacturer’s recommended guidelines and injected on an Applied Biosystems® 3130xl Genetic Analyser at 3kV for 5 and 1 seconds [20]. Analysis and genotyping of data was performed using GeneMapper® ID v3.2.1 software with panels and bins supplied by Promega. Samples were genotyped using internally validated 20 RFU analysis and 100 RFU homozygote thresholds. Overall success rates were determined for full and reduced volume amplifications by calculating the percentage of complete profiles obtained using each method. Success rates were also calculated for the alternate sampling methods (micro-swab versus micro-punch). 3. Results and discussion
Overall success rates determined for both amplification volumes showed little difference, with complete profiles obtained from 80% of samples amplified at reduced volume, compared to 78% at full volume (Table 1). Partial profiles were also very informative, returning a high number of reportable alleles (≥31 alleles from a possible 42 per profile) with the exception of the reduced volume blood on leather micro-punch sample (23 alleles). For this and other blood on non-porous substrate samples (leaf, leather, rubber and latex gloves), more complete profiles were obtained from micro-swabs of the stains rather than micro-punches. Some difficulty was encountered sampling these substrates by micro-punch as blood tended to flake off once dried, making it problematic to capture the desired amount and successfully transfer it to the amplification mixture. The average peak height ratio (PHR) was determined from the heterozygous loci for full and reduced volume amplifications with the variable amounts of input DNA. On average, PHR ranged from 0.81 (D2S1338 and D6S1043) to 0.91 (TH01, vWA and D8S1179) for full volume reactions and 0.79 (D2S1338) to 0.92 (D8S1179) for reduced volume reactions. Other rapid amplification methods have reported PHRs on average above 0.85 [9], and ranging from 0.84
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to 0.92 [8] with ideal amounts of input DNA (≥0.5ng). Inter-locus balance was calculated as the total RFU value of a locus divided by the total RFU value of the profile (for a 21 locus profile each locus would ideally show 0.048 of the total RFU value). Averages ranged from 0.015 (TPOX) to 0.085 (vWA) for full volume reactions and 0.014 (TPOX) to 0.096 (D12S391) for reduced volume reactions. These ranges are also similar to those reported for other rapid methods [16]. Different sized micro-punches (1.2mm for 25µL amplifications and 1mm for 13µL amplifications) were used based on previous in-house optimisation studies for direct amplification of FTA cards which showed a reduction in amplification volume requires a reduction in DNA input amount. Approximately equivalent peak heights are obtained from a 1.0mm micro-punch amplified at 13µL and a 1.2mm micro-punch amplified at 25µL (data not shown). A degree of variation existed in the size of the piece of stained micro-swab material excised for amplification as a result of varying substrate quality and dilution of blood with impurities such as dirt and rust. Typical bloodstain colouration was difficult to distinguish on some of these dirtier swabs; not dissimilar to many casework samples. As a consequence, samples closer to 2mm were taken from these swabs, resulting in noticeably discoloured and coagulated PCR product in some instances (Fig. 2 and Fig. 3C). Results showed this had negligible impact on the ability of PP21 to yield a profile, with the kit demonstrating a robust nature in the presence of noticeable impurities and variable amounts of input DNA. Mock crime scene samples were left to dry for 2 hours, a disparity to actual casework samples where collection would generally occur some hours or days after being deposited, and sometimes much longer. Retrospectively, a longer drying time could have been allowed for these samples to more accurately mimic real casework. Subsequent analysis of casework samples (described following) has shown no disparity between success rates for trial samples and casework samples. As such, it appears, drying time has no effect on success rates. 3.1 Casework application
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Since validation and implementation of this method for amplification of casework blood samples at FSST, 340 samples have been processed. Samples must meet specific requirements to be eligible for direct amplification, namely the stain must be of sufficient size to take additional samples for organic extraction or third party retesting if required, and the presence of blood must be confirmed. For major crime samples the presence of human blood is confirmed using the ABAcard® Hematrace® test kit (Abacus Diagnostics®, part no. 708424) and for minor crime samples the haemochromogen confirmatory test for blood is used [26]. Some improvements to downstream processing have been made to minimise contamination opportunities. Disposable 1mm medical biopsy punches with plunger (Miltex, part no. 33-31AA-P) have been introduced for individual sampling of blood on porous substrates. All samples are placed into individually capped 0.2mL MicroAmp® tubes (Applied Biosystems®, part no. N8010612), stored at -80°C (if required), then carried through the amplification process without the need for sample transfer to a 96-well reaction plate. Blood swabs are sampled as previously described, by cutting a small (1-2mm) piece of the stain and placing it into a labelled 0.2mL MicroAmp® tube (Fig. 3). All samples are amplified with PP21 at reduced volume (13µL), by addition of amplification mix directly into the sample tube and amplification and capillary electrophoresis performed as earlier described. Use of 5 and 1 second injection times enables a greater number of complete profiles to be captured and is necessary given the variable and uncontrollable amounts of DNA being amplified. Introduction of direct amplification and the associated alternative sampling methods has resulted in additional onus on examiners to make an assessment of the suitability of a stain for direct amplification and sample accordingly. After an initial familiarisation period, examiners quickly became comfortable with the sampling method, success rates, the generally variable nature of the stains and optimum sample size, with no issues reported by examiners using the method. Of the 340 samples tested thus far, complete single person profiles were obtained from 70% of samples, partial single person profiles from 8%, mixed profiles from 16%, predominantly dye-blob artefacts from 1%, 1% were overloaded with DNA, and 4% gave no reportable alleles (data not shown). Overall success rates showed 90% of samples yielded an acceptable profile in the first instance and 10% required subsequent organic extraction and qPCR. Acceptable results included some mixed and partial profiles, as partial profiles are generally adequate for matching or exclusionary purposes. During the initial stages of implementation, genotyping was restricted to apparent single person profiles; however standard laboratory mixture interpretation guidelines have now been validated to include genotyping of mixed profiles generated from direct amplification at 27 cycles. Of the 10% (33 samples) to undergo subsequent organic retesting, 31 produced a greater number of reportable alleles. A notable observation has been the negligible effect of stain intensity. Many swabs exhibited pale or dilute bloodstain colouration and complete profiles were still obtained. Similarly good results were obtained from light transfer bloodstains on the surface of clothing where the blood had not penetrated the weave of the fabric to any significant degree. Somewhat surprisingly, the variable nature of casework samples compared to reference samples appears to be of very little consequence.
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Direct amplification is not routinely utilised in-house for its reduced processing time. Samples suitable for direction amplification are stored temporarily and amplified once a small batch of up to 15-30 samples has been filled. Results are then absorbed into standard reporting processes with standard turn around times; reduced processing cost being the main benefit. The true value however, undoubtedly lies where there is an investigative urgency for results from critical samples. Samples are run regardless of batch size and results are provided to police immediately following routine quality assurance checks. For a recent murder case, intelligence level results were provided to detectives in the early stages of the investigation, prior to the interview of suspects. For a recent coronial matter, a priority request was received from the coroners’ office for DNA identification of decomposing human remains, where fingerprint and dental identification were not possible. Due to the level of decomposition, organic extraction of blood from the deceased was attempted in the first instance and yielded no reportable alleles. Given standard extraction timeframes, repeat organic extraction would not normally commence until the following week. Direct amplification was therefore attempted on two more blood samples. Results were obtained by 12pm, with both samples again failing. A sample of spleen from the deceased was then prepared for direct amplification and processed during the afternoon, producing a partial 38 allele profile with a match probability of less than 1 in 100 billion (FSST default). Match details were provided to the coroners’ office on the Friday afternoon, enabling the remains of the deceased to be released to family members without unnecessary delay. The method has also been particularly beneficial for a number of serious assault cases where screening of the victims clothing, heavily stained with the victims own blood, was necessary to identify the presence of blood from the offender. In the above instances, many samples could be taken from bloodstained items and rapidly processed by direct amplification. Processing of suitable casework bloodstains using reduced volume direct amplifications has resulted in nearly a two and a half fold decrease in processing cost per sample. This represents the cost of reagents and consumables and is inclusive of the cost of retesting 10% of samples via the standard organic and qPCR method. The cost of a scientists’ time has not been included, as quantifying these additional savings is difficult to do accurately, although anecdotal evidence suggests a saving of 3 to 4 hours of hands on time. The benefits presented thus far apply broadly in the forensic context regardless of extraction and qPCR chemistries being utilised. Most forensic facilities employ alternative extraction chemistries on automated platforms. These processes are equally as time consuming and costly, and could be bypassed entirely for suitable bloodstains. Increased total laboratory throughput capability is also enabled through the availability of standard extraction places previously occupied by these samples.
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The Promega PowerPlex® 21 PCR Amplification System has demonstrated a clear ability to handle overtly impure casework blood samples and produce complete profiles from direct amplification in less than 3 hours. This has significantly reduced the time and expense required to routinely process samples by removing the requirement for extraction and qPCR. There are obvious benefits to laboratory operations in implementing direct amplification of these sample types, with reduced volume reactions also providing a clear avenue for further fiscal savings. In a non fiscal context, direct amplification has provided significant support for intelligence-led policing and will undoubtedly continue be an advantageous tool in the critical early stages of criminal investigations. Acknowledgements
The authors would like to thank Anna Lemalu for experimental assistance; staff blood donors; and Dr Jason Buchan, Dr Louise McMahon and the technical reviewers for greatly improving this paper through peer review. References
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[12] D.Y. Wang, C-W. Chang, N.J. Oldroyd, L.K. Hennessy, Direct amplification of STRs from blood or buccal cell samples, Forensic Sci. Int. Genet. Suppl. Ser. 2 (2009) 113-114. [13] P.M. Vallone, C.R. Hill, E.L.R. Butts, Concordance study of direct PCR kits: PowerPlex 18D and Identifiler Direct, Forensic Sci. Int. Genet. Suppl. Ser. 3 (2011) e353-e354. [14] B.A. Myers, J.L. King, B. Budowle, Evaluation and comparative analysis of direct amplification of STRs using PowerPlex® 18D and Identifiler® Direct systems, Forensic Sci. Int. Genet. (2012) 640-645. [15] R. Ottens, D. Taylor, D. Abarno, A. Linacre, Successful direct amplification of nuclear markers from a single hair follicle, Forensic Sci. Med. Pathol. 9 (2) (2013) 238-243. [16] S. Verheij, J. Harteveld, T. Sijen, A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples, Forensic Sci. Int. Genet. (2012) 167-175. [17] A. Linacre, V. Pekarek, Y.C. Swaran, S.S. Tobe, Generation of DNA profiles from fabrics without DNA extraction, Forensic Sci. Int. Genet. (2010) 137-141. [18] RapidHIT™ Developmental Validation and Applications Summary, (internet) 2013, (cited: March 7, 2014). Available from: http://integenx.com/wp-content/uploads/2013/09/Developmental-Validation-Data-Summary.pdf [19] M.G. Ensenberger, P.M. Fulmer, B.E. Krenke, R.S. McLaren, C.J. Spreacher, D.R. Storts, Development of the PowerPlex® 21 System, Profiles in DNA (2012) (internet) (cited: September 4, 2013). Available from: http://au.promega.com/resources/articles/profiles-in-dna/2012/development-of-the-powerplex-21-system/
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Table 1 Allele count comparison for all sampling and amplification methods (partial profiles highlighted). A complete PP21 profile has 42 alleles.
Toilet paper Paper towel Tissue Plain white paper Cardboard Satin fabric with interfacing Polyester/elastane fabric A Polyester/elastane fabric B Polyester/elastane fabric (bra) Natural cotton fabric Drill fabric Flannelette fabric Denim fabric Shantung fabric Thermal knit fabric Cotton twill Towelling Woollen beanie Woollen sock Polyester/cotton fabric Chiffon fabric Satin fabric Textile batting Carpet fibres ‘Chux’ cloth Grass Leaf Leather Rubber glove Latex examination glove Press seal plastic bag Plastic supermarket bag Metal spatula Glass Metal bracket Painted wood Copper Bullet Rock Concrete Rusty drain grate Asphalt Garden bark Treated pine Paving stone
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Miscellaneous - non-porous (micro-punch & micro-swab or micro-swab only)
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Fabric/textile - porous (micro-punch only)
Hard - porous (micro-swab only)
% complete profiles
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Paper based - porous (micro-punch only)
25µL amplification 1.2mm Cut piece punch of swab 42 42 37 42 40 42 42 42 42 42 42 42 42 42 42 42 42 42 42 41 41 37 42 42 42 42 42 35 42 41 42 37 42 42 42 42 42 42 42 42 42 41 33 42 42 42 42 42 42 41 73 85 78
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Substrate description
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13µL amplification 1.0mm Cut piece punch of swab 42 42 42 37 33 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 42 31 42 23 42 37 41 37 42 42 42 42 42 42 42 41 32 42 42 42 42 42 42 41 80 80 80
- Indicates sampling method not tested.
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We report on the successful direct amplification of casework blood samples. DNA extraction and quantitation are not required regardless of sample purity. Amplification volume has successfully been reduced. Rapid turn-around times have been achieved in support of intelligence-led policing. Close to a two and a half fold decrease in processing cost has been achieved.
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Additional Files
Fig. 1. Subset of samples tested Fig. 2. 96-well PCR product plate illustrating variable sample colour and degrees of impurity. Numbers indicate allele counts (42 = full profile; control sample allele counts omitted)
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Fig. 3. Mock casework example of direct amplification. (A) dried bloodstain on asphalt (B) micro-swab of bloodstain on asphalt with 1-2mm portion of stain excised from swab head (C) 0.2mL MicroAmp PCR product tube containing excised sample and 13µL of amplification mix (D) Resultant electropherogram (complete profile).
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