Direct amplification of STRs from blood or buccal cell samples

Forensic databasing laboratories routinely analyze blood or buccal cell samples deposited on FTA^® paper. Prior to PCR amplification of the STRs, the FTA^® samples must undergo multi-step sample purification protocols to remove the PCR inhibitors present within the sample and from the FTA^® paper. The multi-step sample purification protocols are laborious, time-consuming and increase the potential for sample cross-contamination.To eliminate the need for DNA purification, we conducted studies to optimize the PCR buffer and thermal cycling parameters to allow for direct amplification of STRs from blood or buccal samples on FTA^® paper. We evaluated the effect of various factors on the DNA profile including: FTA^® disc size, blood sample load variation, and buffer formulation. The new STR assay enables the direct amplification of DNA from single source samples on FTA^® discs without sample purification. The new STR assay improves the workflow by eliminating tedious steps and minimizing sample handling. Furthermore, the new STR assay reduces cost by eliminating the need for purification reagents and expensive robots.     

PowerPlex 16 HS: Internal validation of a new tool for genetic analysis of forensic and parentage testing

Forensic human identification requires powerful and efficient tools to obtain useful results in a minimum timeframe. In this study several forensic and parentage samples that could not be analyzed with others kits were studied using the recently available PowerPlex^® 16 HS kit (Promega). DNA was extracted using four different methods, depending upon the particular sample, and the PCR products were run on an ABI 3130XL Sequencer. The resultant DNA profiles were analyzed using Gene Mapper ID v 3.2 Analysis Software (ABI). Of 30 samples processed with the PowerPlex^® 16 HS system, genetic analysis was successful in 18 (60%). The results obtained show that the PowerPlex^® 16 HS is a valuable tool for forensic identification and parentage testing that is particularly useful for difficult samples that have not yielded adequate results with other methods.     

Development and validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: a multiplex assay for the direct amplification of single-source samples

Abstract: The AmpF?STR^® Identifiler^® Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single‐source blood and buccal samples on FTA^® card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay’s sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA^® cards, and the assay’s specificity was verified by establishing minimal cross‐reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA^® substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler^® Direct Kit for forensic standards and database samples genotyping.     

Performance Testing of a Semi-Automatic Card Punch System,Using Direct STR Profiling of DNA from Blood Samples on FTA™ Cards

The 1.2 mm Electric Coring Tool (e-Core™) was developed to increase the throughput of FTA^™ sample collection cards used during forensic workflows and is similar to a 1.2 mm Harris manual micro-punch for sampling dried blood spots. Direct short tandem repeat (STR) DNA profiling was used to compare samples taken by the e-Core tool with those taken by the manual micro-punch. The performance of the e-Core device was evaluated using a commercially available PowerPlex™ 18D STR System. In addition, an analysis was performed that investigated the potential carryover of DNA via the e-Core punch from one FTA disc to another. This contamination study was carried out using Applied Biosystems AmpflSTR™ Identifiler™ Direct PCR Amplification kits. The e-Core instrument does not contaminate FTA discs when a cleaning punch is used following excision of discs containing samples and generates STR profiles that are comparable to those generated by the manual micro-punch.     

AmpFlSTR MiniFiler™ PCR amplification kit: The new miniSTR multiplex kit

The AmpFlSTR^® MiniFiler™ PCR amplification kit (Applied Biosystems), a new available 8-miniSTR and the sex determining marker Amelogenin multiplex, includes the most common problematic loci (above 200 bp) of the AmpFlSTR^® Identifiler™ PCR amplification kit: FGA, D21S11, D18S51, D13S317, D7S820, D16S539, CSF1PO and D2S1338.Several casework samples with different DNA contents were tested.Results allowed to complete partial Identifiler™ profiles and additional information was achieved in low copy number (LCN) samples, revealing that this miniSTR kit can improve identification of compromised samples.     

Successful STR and SNP typing of FTA Card samples with low amounts of DNA after DNA extraction using a Qiagen BioRobot EZ1 Workstation

FTA Cards (GE Healthcare) have been used for more than 4 years in Denmark for the collection of buccal cells as reference samples in crime cases. Semi-automated protocols for STR typing of DNA on punches of FTA Cards are routinely used. In average, full STR profiles were generated from approximately 95% of the FTA Cards with a standard punching protocol, while partial or no STR profile were obtained from 5% of the samples. Here, the Qiagen BioRobot^® EZ1 Workstation (Qiagen) and the EZ1 DNA Investigator Kit (Qiagen) was used to extract DNA from 29 FTA Cards from which a complete STR profile was not generated with the standard punching protocol. All 29 samples were successfully typed with the AmpF?STR^® Identifiler™ PCR Amplification Kit (Applied Biosystems) and with the SNPforID 49plex SNP assay. The lowest amount of DNA that resulted in complete STR and SNP profiles was 80 pg. The STR and SNP profiles were identical to those generated from another sample collected from each of the 29 individuals.     

A dedicated automated system for extraction, quantification and STR amplification of forensic evidence samples

Forensic DNA analysis is a multi-step process involving extraction of DNA, quantification of human DNA in the extract, amplification using multiplex STR systems, separation of products, and data analysis. The backlog of forensic casework is increasing worldwide. Automation is one significant way to alleviate the bottleneck of sample processing in forensic labs. The HID EVOlution™ Combination System described here is a robust, reliable sample processing platform, easily adapted to forensic laboratory workflows. Using a variety of forensic sample types including: blood stained FTA paper, cotton fabric and denim, dried blood spiked with known PCR inhibitors, saliva on cotton swabs, and semen stains, we found that yields of human DNA and STR profiles obtained with AmpFlSTR^® Idenitfiler^® kits were complete, highly reproducible, and equivalent to results obtained using the manual PrepFiler™ reagent extraction method. Automated operation was clean, and no cross-contamination was detected between extraction blanks and interspersed high DNA content samples.     

Optimization of DNA recovery from toothbrushes

In human identification, the victim's toothbrush is an invaluable personal item as the deposited cellular material contains DNA from which a reference profile can be produced. The profile obtained then allows direct comparison to be made with the profile from the unidentified body. This study was undertaken to determine the minimum number of bristle bundles that would generate a complete DNA profile. The minimum period of usage for a toothbrush to retain enough cells for genotyping was also investigated. We also tested two commonly used DNA extraction methods: QIAamp^® DNA Mini Kit and Chelex^® 100 to explore the efficiency of these protocols in recovering DNA from toothbrushes. In this experiment, volunteers brushed their teeth for 1, 7, 14, or 30 days. DNA was extracted from 5 and 10 bundles of bristles cut from the collected toothbrushes. The amount of DNA recovered was quantified by quantitative real-time PCR, and DNA genotyping was performed for each sample. Data revealed that QIAamp^® DNA Mini Kit performed better at yielding DNA in terms of purity, quantity, and quality than Chelex^® 100. It was also found that, with a suitable method of recovery, DNA samples from five bundles of bristles from all of the toothbrushes generated complete profiles. Based on the experimental results, a general guideline concerning the appropriate extraction method and the quantity of the starting material for the analysis of DNA from toothbrushes could be suggested.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.     

Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures

Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100 pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpF?STR^® Identifiler^® amplification parameters are changed to an annealing time of 20 min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36 pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpF?STR^® Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency.     

Generating DNA profiles from immunochromatographic cards using LCN methodology

The aim of this research was to obtain DNA profiles from immunochromatographic test devices which have already yielded positive results with body fluids obtained from fourteen volunteers. Three different immunochromatographic cards for the identification of human blood and one for the identification of human saliva were used for this research. Each body fluid was detected using the appropriate immunochromatographic card. The used cards were kept at room temperature for various lengths of time. The membranes were removed at the end of the designated times and the entire strip was extracted using low copy number (LCN) extraction procedure. The extracted DNA was amplified using reduced amplification volume and higher PCR cycle numbers. Autosomal STR profiles were detected using AmpFℓSTR^® Identifiler™ PCR Amplification Kit from Applied Biosystems (AB). Additionally, DNA extracted from the male volunteers was amplified using the AB AmpFℓSTR^® Yfiler™ PCR Amplification Kit. Analysis of the amplified products was carried out by capillary electrophoresis injection on the AB 3130xl Genetic Analyzer. The generated DNA data was analyzed using the SoftGenetics GeneMarker^® HID Version 1.7 software.Autosomal and Y-STR DNA profiles were obtained from most of the cards which were stored at room temperature for up to three months. DNA profile was obtained from all four types of the immunochromatographic cards used in this study. These profiles were concordant with the profiles obtained from the donors’ reference samples.     

Optimization and validation of a fast amplification protocol for AmpFlSTR Profiler Plus for rapid forensic human identification

The goal of this work was to optimize and validate a fast amplification protocol for the multiplex amplification of the STR loci included in AmpFlSTR^® Profiler Plus^® to expedite human DNA identification. By modifying the cycling conditions and by combining the use of a DNA polymerase optimized for high speed PCR (SpeedSTAR™ HS) and a more efficient thermal cycler instrument (Bio-RAD C1000™), we were able to reduce the amplification process from 4 h to 26 min. No modification to the commercial AmpFlSTR^® Profiler Plus^® primer mix was required. When compared to the current Royal Canadian Mounted Police (RCMP) amplification protocol, no differences with regards to specificity, sensitivity, heterozygote peak height ratios and overall profile balance were noted. Moreover, complete concordance was obtained with profiles previously generated with the standard amplification protocol and minor alleles in mixture samples were reliably typed. An increase in n − 4 stutter ratios (2.2% on average for all loci) was observed for profiles amplified with the fast protocol compared to the current procedure. Our results document the robustness of this rapid amplification protocol for STR profiling using the AmpFlSTR^® Profiler Plus^® primer set and demonstrate that comparable data can be obtained in substantially less time. This new approach could provide an alternative option to current multiplex STR typing amplification protocols in order to increase throughput or expedite time-sensitive cases.     

A novel platform for the modular integration of forensic assay setup and medium- to high-throughput purification of nucleic acids

With its extraction and assay setup modules, the QIAsymphony^® provides a highly flexible solution for processing forensic samples in a medium- to high-throughput scale. We tested the sensitivity of extraction, precision of sample processing, and accuracy of automated assay setup of this integrated system. Results attest to QIAsymphony's ability to isolate DNA from a spectrum of common forensic samples and process these samples without cross-contamination. Furthermore, accurate assay setup for downstream applications, like PCR, make this system highly suited for enhancing laboratory workflow.     

A fast analysis system for forensic DNA reference samples

On January 1st, 2006, the Swedish legislation on obtaining DNA reference samples from suspects and the recording of DNA profiles in databases was changed. As a result the number of samples analysed at the Swedish National Laboratory of Forensic Science (SKL) increased from about 4500 in 2005 to more than 25,000 in 2006. To meet this challenge, SKL launched a new analysis system to create an unbroken chain, from sampling to incorporation of a profile in the national DNA database and subsequent automatic generation of digitally signed hit reports. The system integrates logistics, digital data transfer, new functions in LIMS (ForumDNA Version 4, Ida Infront AB) and laboratory automation. Buccal swab samples are secured on a FTA^® card attached to an identity form, which is barcoded with a unique sample ID. After sampling, the police officer sends a digital request to SKL. The sample is automatically registered in LIMS and processed on delivery. The resulting DNA profiles are automatically classified according to quality using a custom-made expert system. Building the evaluation around mathematical rules makes it reproducible, standardised and minimises manual work and clerk errors. All samples are run in duplicate and the two profiles are compared within LIMS before incorporation in the database. In the first year of operation, the median time for completion of an analysis was 3 days, measured from delivery of the sample to incorporation of the profile in the national DNA database. In spite of the dramatic increase in the number of reference samples there was no backlog.     

Extraction of high quality DNA from biological materials and calcified tissues

Calcified tissues, such as bone and tooth, and some other sample types, such as those containing adhesive, present a challenge to standard extraction protocols. We have developed a lysis reagent, BTA™ lysis buffer, which is designed for use with PrepFiler™ Kit reagents. The BTA™ lysis buffer disrupts calcified tissue matrices and achieves effective extraction of DNA from pulverized bone and tooth samples. In addition, the BTA™ lysis buffer mildly but efficiently extracts DNA from challenging substrates like tape, chewing gum, and cigarette butts and, as with bone and tooth, DNA from these lysates is purified using established PrepFiler™ reagent extraction protocols.We successfully extracted DNA from powdered human bone samples, chewed gum and smoked cigarettes using BTA™ lysis buffer. Extraction yields for bone, gum and cigarette samples tested were consistent and reproducible. This extraction method efficiently removed potential PCR inhibitors from all samples tested, and C_T values for the internal PCR control of Quantifiler^® Human DNA Quantification Kit were consistent and within the normal range. The DNA extracted from these samples also provided conclusive profiles that were free of PCR artifacts when amplified using the AmpF?STR^® Identifiler^® PCR Amplification Kit. The protocol is easily adapted for automation.     

Comparisons of protocols for enhanced DNA profile quality from FTA®-deposited urine samples

Disputes over the identity of a urine sample donor have been reported, and urine authentication by genetic profiling has helped resolved the cases. However, since genotyping of urine is not always required, many drug-testing laboratories may face sample storage issues. Several studies have investigated the use of FTA® cards as a convenient tool for keeping specimen at room temperature for extended periods of time. However, generating complete STR profile from some FTA®-deposited urine samples remains challenging due to low levels of genetic material content, necessitating amendments to the laboratory’s standard protocols. This work therefore aims to evaluate the effects of two DNA template preparation methods, both employing FTA® cards as the storage medium, on the success rates of STR profiling from urine. Specimen from a female volunteer, representing a particularly low-yield sample, was employed. Aliquots of 1 and 2 mL were used as the starting material to evaluate DNA template preparation using the FTA® manufacturer’s protocol for disc purification against elution of DNA from the FTA® using Prepfiler™ Forensic DNA Extraction Kit. AmpFℓSTR™ Identifiler™ Plus PCR Amplification Kit was used to amplify the STR markers, and the PCR products were analysed using Applied Biosystems™ 3500xL Genetic Analyzer. The DNA profile qualities were examined in terms of number of loci detected and peak height balance. Comparisons with the profiles obtained from DNA isolated using QIAamp® DNA Micro Kit from 1 and 2 mL of the same batch of urine were also made. The optimised protocol was then tested on urine samples from three male volunteers. The results showed that the purification of FTA® punches according to the manufacturer’s protocol enabled full DNA profiles to be obtained from both 1 and 2 mL of urine from all samples tested, including male samples. In contrast, no DNA profile could be generated from the DNA eluted with the Prepfiler™ kit. When compared with the more conventional solid-phase DNA extraction method, the profiles generated from the FTA® punches exhibited similar reproducibility and quality to those from the template isolated by the QIAamp® Kit. This work further demonstrated the feasibility of FTA® cards as a tool for specimen storage and DNA template preparation from small volumes of urine for authentication by STR profiling. Full STR profiles could be generated from sample from both sexes without modification of the PCR conditions or injection time.     

Automated extraction of DNA from reference samples from various types of biological materials on the Qiagen BioRobot EZ1 Workstation

We have validated and implemented a protocol for DNA extraction from various types of biological materials using a Qiagen BioRobot EZ1 Workstation. The sample materials included whole blood, blood from deceased, buccal cells on Omni swabs and FTA Cards, blood on FTA Cards and cotton swabs, and muscle biopsies. The DNA extraction was validated according to EN/ISO 17025 for the STR kits AmpF?STR^® Identifiler^® and AmpF?STR^® Yfiler^® (Applied Biosystems). Of 298 samples extracted, 11 (4%) did not yield acceptable results. In conclusion, we have demonstrated that extraction of DNA from various types of biological material can be performed quickly and without the use of hazardous chemicals, and that the DNA may be successfully STR typed according to the requirements of forensic genetic investigations accredited according to EN/ISO 17025.     

The use of DNA stabilizing solution to enable room temperature storage and transportation of buccal and trace sample swabs

It is proposed that a DNA stabilizing solution (DNA Genotek Inc.) designed to preserve DNA in saliva samples at room temperature can be extrapolated to the storage of swab heads. The aim of this study was to evaluate the effectiveness of the solution for the preservation of reference swabs (buccal) and trace samples (facial swabs). To this end, the solution was used during a twin-site DNA transfer project assessing background levels of carer DNA present in children. Tubes containing 400 μl of solution were used to store and transport swab heads. At the laboratory, samples were extracted using the QIAamp DNA Mini Kit (Qiagen), quantified using the Quantifiler Duo Kit and profiled using the AmpF?STR^® SGM Plus^® PCR Amplification Kit (both Applied Biosystems). Twenty-eight PCR cycles were applied to all samples. Thirty-four cycles or a longer electrophoresis injection time was applied to trace samples where necessary. All Reference swabs produced high quantities of DNA and full DNA profiles after 28 cycles. Profile morphology indicated good quality DNA with no degradation. Of the trace samples, sufficient profiles were achieved to study the transfer of carer DNA making the solution fit for continued use in this project. DNA stabilizing solution enables the storage and transportation of swabs without freezing. This is convenient, reduces transportation costs and enables instant analysis of samples upon arrival at the laboratory. This is a useful alternative for a multi-site research project as well as a reliable storage tool for use in remote areas.     

Simplified low-copy-number DNA analysis by post-PCR purification

Frequently, evidentiary items contain an insufficient quantity of DNA to obtain complete or even partial DNA profiles using standard forensic gentotyping techniques. Such low-copy-number (LCN) samples are usually subjected to increased amplification cylces to obtain genetic data. In this study, a 28-cycle polymerase chain reaction (PCR) was used to evaluate various methods of post-PCR purification for their effects on the sensitivity of fluorophore-based allelic detection subsequent to capillary electrophoretic separation. The amplified product was purified using filtration, silica gel membrane, and enzyme mediated hydrolysis purification techniques and evaluated for their effect on fluorescent allelic signal intensity. A purification method was selected and its effect on fluorescent allelic signal intensity was compared with that of the unpurified PCR product. A method of post-PCR purification is described which increases the sensitivity of standard 28-cycle PCR such that profiles from LCN DNA templates (<100 pg DNA) can be obtained. Full DNA profiles were consistently obtained with as little as 20 pg template DNA without increased cycle number. In mock case type samples with dermal ridge fingerprints, genetic profiles were obtained by amplification with 28 cycles followed by post-PCR purification whereas no profiles were obtained without purification of the PCR product. Allele dropout, increased stutter, and sporadic contamination typical of LCN analysis were observed; however, no contamination was observed in negative amplification controls. Post-PCR purification of the PCR product can increase the sensitivity of capillary electrophoresis to such an extent that DNA profiles can be obtained from <100 pg of DNA using 28-cycle amplification.     

Automated Quantifiler quantitative PCR setup, template normalization and PCR setup using HID EVOlution™ qPCR/STR setup on trace evidence samples

We have implemented and validated customized protocols for automated Quantifiler^® setup, template normalization and PCR setup using the Tecan HID EVOlution™ qPCR/STR setup. The protocols were validated for the Quantifiler^® human DNA quantification, AmpF?STR^® SGM Plus^® and SEfiler Plus™ PCR Amplification Kits (Applied Biosystems) according to EN/ISO 17025.