A comparison of four methods for PCR inhibitor removal

Biological samples collected from the crime scenes often contain some compounds that can inhibit the polymerase chain reaction (PCR). The removal of PCR inhibitors from the extracts prior to the PCR amplification is vital for successful forensic DNA typing. This paper aimed to evaluate the ability of four different methods (PowerClean^® DNA Clean-Up kit, DNA IQ™ System, Phenol–Chloroform extraction and Chelex^®-100 methods) to remove eight commonly encountered PCR inhibitors including: melanin, humic acid, collagen, bile salt, hematin, calcium ions, indigo and urea. Each of these PCR inhibitors was effectively removed by the PowerClean^® DNA Clean-Up kit and DNA IQ™ System as demonstrated by generating more complete short tandem repeat (STR) profiles from the cleaned up inhibitor samples than from the raw inhibitor samples. The Phenol–Chloroform extraction and Chelex^®-100 methods, however, could only remove some of eight PCR inhibitors. Our results demonstrated that the PowerClean^® DNA Clean-Up kit and DNA IQ™ System were very effective for the removal of known PCR inhibitors that are routinely found in DNA extracts from forensic samples.     

Stochastic sampling effects in STR typing: Implications for analysis and interpretation

The analysis and interpretation of forensic STR typing results can become more complicated when reduced template amounts are used for PCR amplification due to increased stochastic effects. These effects are typically observed as reduced heterozygous peak-height balance and increased frequency of undetected alleles (allelic “dropout”). To investigate the origins of these effects, a study was performed using the AmpFlSTR^® Identifiler Plus^® and MiniFiler^® kits to amplify replicates from a dilution series of NIST Human DNA Quantitation Standard (SRM^® 2372A). The resulting amplicons were resolved and detected on two different genetic analyzer platforms, the Applied Biosystems 3130xL and 3500 analyzers. Results from our study show that the four different STR/genetic analyzer combinations exhibited very similar peak-height ratio statistics when normalized for the amount of template DNA in the PCR. Peak-height ratio statistics were successfully modeled using the Poisson distribution to simulate pre-PCR stochastic sampling of the alleles, confirming earlier explanations that sampling is the primary source for peak-height imbalance in reduced template dilutions. In addition, template-based pre-PCR sampling simulations also successfully predicted allelic dropout frequencies, as modeled by logistic regression methods, for the low-template DNA dilutions. We discuss the possibility that an accurately quantified DNA template might be used to characterize the linear signal response for data collected using different STR kits or genetic analyzer platforms, so as to provide a standardized approach for comparing results obtained from different STR/CE combinations and to aid in validation studies.     

PCR inhibitor removal using the NucleoSpin® DNA Clean-Up XS kit

Forensic evidence samples are collected from an unlimited variety of substrates, which may contain compounds known to inhibit the polymerase chain reaction (PCR). These PCR inhibitors are co-extracted with the DNA sample and can negatively affect the DNA typing results, which can range from partial to complete inhibition of the short tandem repeat (STR) PCR. One potential solution is to remove the PCR inhibitors from the extracts prior to the STR PCR with the NucleoSpin^® DNA Clean-Up XS kit. The kit contains a NucleoSpin^® XS silica column that has a special funnel design of thrust rings along with a very small silica membrane, which allows for sample elution in a small volume that is appropriate for use with current STR typing kits. The NucleoSpin^® DNA Clean-Up XS kit was optimized for the best possible DNA recovery and then evaluated for its ability to remove eight commonly encountered PCR inhibitors including: bile salt, collagen, hematin, humic acid, indigo, melanin, tannic acid and urea. Each of these PCR inhibitors was effectively removed by the NucleoSpin^® DNA Clean-Up XS kit as demonstrated by generating more complete STR profiles from the cleaned up inhibitor samples than from the raw inhibitor samples.     

Quantifiler® Trio Kit and forensic samples management: A matter of degradation

DNA collected from crime scenes may have experienced different levels of degradation. This is mainly due to sample exposure to different environmental factors. The impact of DNA degradation on short tandem repeat (STR) profiling can lead to partial or null information and in some cases, the identification of the trace may fail. The availability of a system enabling the assessment not only of the quantity of the DNA but also of its quality in terms of degradation would result in shorter time for sample processing, more reliable identifications and cost reduction by predicting the quality of the DNA profiles prior to STR analysis. We report here a study on 181 selected degraded DNA samples extracted from real crime scene evidence. The selected samples were processed by combining the use of a new commercial quantification kit (Quantifiler^® Trio) with a new 24 marker multiplex PCR amplification kit (Globalfiler^® Kit). Applying different statistical analyses we investigated the reliability of the Degradation Index provided by the Quantifiler^® Trio in determining the level of DNA degradation in a forensic sample. This useful information can be used to predict the quality of the profile obtained after STR amplification. The combination of such a quantification kit with different PCR protocols allowed us to define practical guidelines for processing degraded forensic DNA samples with a simplified and comprehensive approach.     

Comparative evaluation of different extraction and quantification methods for forensic RNA analysis

Since about 2005, there is increasing interest in forensic RNA analysis whose versatility may very favorably complement traditional DNA profiling in forensic casework. There is, however, no method available specifically dedicated for extraction of RNA from forensically relevant sample material.In this study we compared five commercially available and commonly used RNA extraction kits and methods (mirVana™ miRNA Isolation Kit Ambion; Trizol^® Reagent, Invitrogen; NucleoSpin^® miRNA Kit Macherey-Nagel; AllPrep DNA/RNA Mini Kit and RNeasy^® Mini Kit both Qiagen) to assess their relative effectiveness of yielding RNA of good quality and their compatibility with co-extraction of DNA amenable to STR profiling.We set up samples of small amounts of dried blood, liquid saliva, semen and buccal mucosa that were aged for different time intervals for co-extraction of RNA and DNA. RNA quality was assessed by determination of ‘RNA integrity number’ (RIN) and quantitative PCR based expression analysis. DNA quality was assessed via monitoring STR typing success rates.By comparison, the different methods exhibited considerable differences between RNA and DNA yields, RNA quality values and expression levels, and STR profiling success, with the AllPrep DNA/RNA Mini Kit and the NucleoSpin^® miRNA Kit excelling at DNA co-extraction and RNA results, respectively.Overall, there was no ‘best’ method to satisfy all demands of comprehensible co-analysis of RNA and DNA and it appears that each method has specific merits and flaws. We recommend to cautiously choose from available methods and align its characteristics with the needs of the experimental setting at hand.     

Developmental validation of a fully integrated sample-to-profile rapid human identification system for processing single-source reference buccal samples

Short tandem repeat (STR) DNA typing is a global standard for human identification. Current practice involves highly trained forensic analysts, operating in a laboratory setting, using multiple instruments to process samples and analyze the data. Here, we report the developmental validation of a fully integrated and automated DNA profiling system, the RapidHIT^® System, capable of producing up to five high quality STR profiles with full controls in approximately 90 min using PowerPlex^®16 HS RapidHIT chemistry. The system integrates all sample handling steps: starting from lysis of cells on buccal swabs or other buccal sample types through DNA extraction, normalization, amplification,capillary array electrophoresis, detection, and integrated software analysis.The results describe the developmental validation of the RapidHIT™ System for buccal samples processed with the DNA IQ™ extraction chemistry using a guandinium chaotropic agent and paramagnetic beads followed by amplification using a modified version of PowerPlex 16 HS chemistry (PowerPlex 16 HS RapidHIT chemistry), and capillary electrophoresis with manual review of genotyping data following interpretation guidelines. All processing from the buccal swab to generation and processing of the profile occurs on the RapidHIT platform.Resultare concordant with traditional methods, with 88% first pass success rates for both the CODIS and PowerPlex 16 loci. Average peak height ratios were 0.89 for buccal swabs. The system produces full profiles from swabs with at least 176 ng of saliva DNA. Rapid DNA identification systems will significantly enhance capabilities for forensic labs, intelligence, defense, law enforcement, refugee and immigration applications, and kinship analysis.     

The Qiagen Investigator® Quantiplex HYres as an alternative kit for DNA quantification

The Investigator^® Quantiplex HYres kit was evaluated as a potential replacement for dual DNA quantification of casework samples. This kit was determined to be highly sensitive with a limit of quantification and limit of detection of 0.0049 ng/μL and 0.0003 ng/μL, respectively, for both human and male DNA, using full or half reaction volumes. It was also accurate in assessing the amount of male DNA present in 96 mock and actual casework male:female mixtures (various ratios) processed in this exercise. The close correlation between the male/human DNA ratios expressed in percentages derived from the Investigator^® Quantiplex HYres quantification results and the male DNA proportion calculated in mixed AmpFlSTR^® Profiler^® Plus or AmpFlSTR^® Identifiler^® Plus profiles, using the Amelogenin Y peak and STR loci, allowed guidelines to be developed to facilitate decisions regarding when to submit samples to Y-STR rather than autosomal STR profiling. The internal control (IC) target was shown to be more sensitive to inhibitors compared to the human and male DNA targets included in the Investigator^® Quantiplex HYres kit serving as a good quality assessor of DNA extracts. The new kit met our criteria of enhanced sensitivity, accuracy, consistency, reliability and robustness for casework DNA quantification.     

Concordance study between the ParaDNA® Intelligence Test,a Rapid DNA profiling assay,and a conventional STR typing kit (AmpFlSTR® SGM Plus®)

The ParaDNA^® Intelligence Test enables STR profiling directly from human biological samples and evidence items collected from crime scene in 75 min. Designed for non-expert use this system allows DNA information to be available to investigators before it would typically be available from a laboratory. The ParaDNA Intelligence Test system amplifies D3S1358, D8S119, D16S539, D18S1358 and TH01 STR loci and the gender typing locus amelogenin and detects the alleles present with HyBeacon^® probes. Individual DNA samples from 381 UK Caucasian individuals were analysed using AmpFlSTR^® SGM Plus^® and the ParaDNA Intelligence Test with the derived STR profiles compared. Here we describe the high level of concordance demonstrated between the two systems and discuss this with reference to allele frequencies and the discriminatory power offered by the ParaDNA Intelligence Test.     

Developmental validation of the ParaDNA® Intelligence System—A novel approach to DNA profiling

DNA profiling through the analysis of STRs remains one of the most widely used tools in human identification across the world. Current laboratory STR analysis is slow, costly and requires expert users and interpretation which can lead to instances of delayed investigations or non-testing of evidence on budget grounds. The ParaDNA^® Intelligence System has been designed to provide a simple, fast and robust way to profile DNA samples in a lab or field-deployable manner. The system analyses 5-STRs plus amelogenin to deliver a DNA profile that enables users to gain rapid investigative leads and intelligent prioritisation of samples in human identity testing applications. Utilising an innovative sample collector, minimal training is required to enable both DNA analysts and nonspecialist personnel to analyse biological samples directly, without prior processing, in approximately 75 min. The test uses direct PCR with fluorescent HyBeacon^® detection of STR allele lengths to provide a DNA profile. The developmental validation study described here followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines and tested the sensitivity, reproducibility, accuracy, inhibitor tolerance, and performance of the ParaDNA Intelligence System on a range of mock evidence items. The data collected demonstrate that the ParaDNA Intelligence System displays useful DNA profiles when sampling a variety of evidence items including blood, saliva, semen and touch DNA items indicating the potential to benefit a number of applications in fields such as forensic, military and disaster victim identification (DVI).     

Null alleles and sequence variations at primer binding sites of STR loci within multiplex typing systems

Rare variants are widely observed in human genome and sequence variations at primer binding sites might impair the process of PCR amplification resulting in dropouts of alleles, named as null alleles. In this study, 5 cases from routine paternity testing using PowerPlex^®21 System for STR genotyping were considered to harbor null alleles at TH01, FGA, D5S818, D8S1179, and D16S539, respectively. The dropout of alleles was confirmed by using alternative commercial kits AGCU Expressmarker 22 PCR amplification kit and AmpFℓSTR^®. Identifiler^® Plus Kit, and sequencing results revealed a single base variation at the primer binding site of each STR locus. Results from the collection of previous reports show that null alleles at D5S818 were frequently observed in population detected by two PowerPlex^® typing systems and null alleles at D19S433 were mostly observed in Japanese population detected by two AmpFℓSTR™ typing systems. Furthermore, the most popular mutation type appeared the transition from C to T with G to A, which might have a potential relationship with DNA methylation. Altogether, these results can provide helpful information in forensic practice to the elimination of genotyping discrepancy and the development of primer sets.     

A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples

Forensic DNA typing involves a multi-step workflow. Steps include DNA isolation, quantification, amplification of a set of short tandem repeat (STR) markers, separation of polymerase chain reaction (PCR) products by capillary electrophoresis (CE) and DNA profile analysis and interpretation. With that, the process takes around 10–12 h. For several scenarios it may be very valuable to speed up this process and obtain an interpretable DNA profile, suited to search a DNA database, within a few hours. For instance in cases of national security, abduction with danger of life, risk of repetition by a serial perpetrator or when custody time of suspects is limited. By a direct and rapid PCR approach we reduced the total DNA profiling time to 2–3 h after which genotyping information for the 10 STR markers plus the amelogenin (AMEL) marker present in the commercially available AmpF?STR^® SGM Plus? (SGM+) profiling kit is obtained. This reduction in time is achieved by using the following elements: (1) the inhibitor tolerant, highly processive Phusion^® Flash DNA polymerase; (2) a modified, non-adenylated allelic ladder; (3) the quick PIKO^® thermal cycler system with ultra-thin walled reaction tubes; (4) profile interpretation guidelines with an increased allele calling threshold, modified stutter ratios and marked low-level artefact peaks and (5) regulation of sample input by the use of mini-tapes that lift a limited amount of cell material from swabs or fabrics. The procedure is specifically effective for high level DNA, single source samples such as samples containing saliva, blood, semen and hair roots. Success rates, defined as a complete DNA profile, depend on stain type and surface. Due to the use of tape lifting as the sampling technique, the swab or fabric remains dry and intact and can be analyzed at a later stage using regular procedures. Validation experiments were performed which showed that the protocol effectively instructs researchers unfamiliar with the procedure. We have incorporated direct and rapid PCR in a “DNA-6 h” service that can assist police investigations by rapidly deriving DNA information from trace evidence left by a perpetrator, searching the STR profile against a DNA database and reporting the outcomes to police or prosecution.     

Ein DNS-Extraktionskit „für alle Fälle“?

The DNA extraction kit GEN-IAL^®-First DNA (GEN-IAL^®, Troisdorf, Germany), first produced to isolate DNA from plants and food, was validated for forensic material. A large panel of human material was tested: blood, buccal swabs, chewing gum, urine, semen, frozen tissue, paraffin-embedded tissue, labelled and fixed tissue sections, bone, hair roots and hair shafts. The success of DNA isolation was controlled by PCR. DNA could be extracted from all materials tested. A quantitative PCR analysis (LightCycler^®; Roche Diagnostics, Mannheim, Germany) was done using the DNA extracted from frozen tissue and from hair roots. In this assay the GEN-IAL^®- kit gave better results concerning the quality and quantity of the extracted DNA than DNA isolated with the QIAamp kit (QIAGEN, Hilden, Germany) and the phenol extraction method. Together with the materials already tested (e.g. cell cultures, plants, bacteria, yeast, food) the GEN-IAL^®-kit seems to be a universally applicable DNA extraction method suitable for practically all relevant substrates for diagnostic and research purposes.     

An evaluation of the RapidHIT® system for reliably genotyping reference samples

Short tandem repeat (STR) typing is used routinely for associating or excluding individuals with biological evidence left at a crime scene. Improvements have been made to reduce the turnaround time and labor involved with profile generation, but there is still some lag time between sample collection and interpretation of results. The RapidHIT^® (IntegenX; Pleasanton, CA, USA) system is an automated instrument that is configured to perform DNA extraction, bead-based DNA normalization, amplification, electrophoresis of PCR amplicons, and data analysis of five reference swabs simultaneously. The RapidHIT system provided reliable STR profiles from reference buccal swabs in approximately 90 min with nominal “hands-on” sample loading time with no evidence of contamination between samples. The overall success rate of typing buccal swabs was comparable to standard typing systems. In the event of a failed run due to instrument failure, the swab can be removed from the cartridge and reanalyzed in the RapidHIT system or with standard STR genotyping workflows.     

The effects of metal ion PCR inhibitors on results obtained with the Quantifiler® Human DNA Quantification Kit

Forensic DNA samples may include the presence of PCR inhibitors, even after extraction and purification. Studies have demonstrated that metal ions, co-purified at specific concentrations, inhibit DNA amplifications. Metal ions are endogenous to sample types, such as bone, and can be introduced from environmental sources. In order to examine the effect of metal ions as PCR inhibitors during quantitative real-time PCR, 2800 M DNA was treated with 0.0025–18.750 mM concentrations of aluminum, calcium, copper, iron, nickel, and lead. DNA samples, both untreated and metal-treated, were quantified using the Quantifiler^® Human DNA Quantification Kit. Quantification cycle (Cq) values for the Quantifiler^® Human DNA and internal PCR control (IPC) assays were measured and the estimated concentrations of human DNA were obtained. Comparisons were conducted between metal-treated and control DNA samples to determine the accuracy of the quantification estimates and to test the efficacy of the IPC inhibition detection. This kit is most resistant to the presence of calcium as compared to all metals tested; the maximum concentration tested does not affect the amplification of the IPC or quantification of the sample. This kit is most sensitive to the presence of aluminum; concentrations greater than 0.0750 mM negatively affected the quantification, although the IPC assay accurately assessed the presence of PCR inhibition. The Quantifiler^® Human DNA Quantification Kit accurately quantifies human DNA in the presence of 0.5000 mM copper, iron, nickel, and lead; however, the IPC does not indicate the presence of PCR inhibition at this concentration of these metals. Unexpectedly, estimates of DNA quantity in samples treated with 18.750 mM copper yielded values in excess of the actual concentration of DNA in the samples; fluorescence spectroscopy experiments indicated this increase was not a direct interaction between the copper metal and 6-FAM dye used to label the probe that targets human DNA in the Quantifiler^® kit. Evidence of inhibition was observed for the human-specific assay at a lower metal concentration than detected by the IPC, for all metals examined except calcium. These results strongly suggest that determination of a “true negative” sample should not be based solely on the failure of the IPC to indicate the presence of a PCR inhibitor and indicate that amplification of all samples should be attempted, regardless of the quantification results.     

Population genetic analyses of the STR loci of the AmpFlSTR NGM SElect™ kit for Han population in Fujian Province, China

Allele frequencies and forensically relevant population statistics of the STR loci in the AmpFlSTR® NGM SElect? PCR Amplification Kit were estimated for the Han population from Fujian province in China (n?=?454). All loci were highly polymorphic and the cumulative match probability was 5.4?×?10^?21. No significant departure from Hardy–Weinberg equilibrium and linkage equilibrium was detected after correction for sampling. The population substructure of Fujian Han population is minor.     

Comparing different post-mortem human samples as DNA sources for downstream genotyping and identification

The capability of DNA laboratories to perform genotyping procedures from post-mortem remains, including those that had undergone putrefaction, continues to be a challenge in the Philippines, a country characterized by very humid and warm conditions all year round. These environmental conditions accelerate the decomposition of human remains that were recovered after a disaster and those that were left abandoned after a crime. When considerable tissue decomposition of human remains has taken place, there is no other option but to extract DNA from bone and/or teeth samples. Routinely, femur shafts are obtained from recovered bodies for human identification because the calcium matrix protects the DNA contained in the osteocytes. In the Philippines, there is difficulty in collecting femur samples after natural disasters or even human-made disasters, because these events are usually characterized by a large number of fatalities. Identification of casualties is further delayed by limitation in human and material resources. Hence, it is imperative to test other types of biological samples that are easier to collect, transport, process and store.We analyzed DNA that were obtained from body fluid, bone marrow, muscle tissue, clavicle, femur, metatarsal, patella, rib and vertebral samples from five recently deceased untreated male cadavers and seven male human remains that were embalmed, buried for ∼1 month and then exhumed. The bodies had undergone different environmental conditions and were in various stages of putrefaction. A DNA extraction method utilizing a detergent-washing step followed by an organic procedure was used. The utility of bone marrow and vitreous fluid including bone marrow and vitreous fluid that was transferred on FTA^® cards and subjected to autosomal STR and Y-STR DNA typing were also evaluated. DNA yield was measured and the presence or absence of PCR inhibitors in DNA extracts was assessed using Plexor^®HY. All samples were amplified using PowerPlex^®21 and PowerPlexY^®23 systems and analyzed using the AB3500 Genetic Analyzer and the GeneMapper^® ID-X v.1.2 software.PCR inhibitors were consistently detected in bone marrow, muscle tissue, rib and vertebra samples. Amplifiable DNA was obtained in a majority of the samples analyzed. DNA recovery from 0.1 g biological material was adequate for successful genotyping of most of the non-bone and bone samples. Complete DNA profiles were generated from bone marrow, femur, metatarsal and patella with 0.1 ng DNA template. Using 0.5 ng DNA template resulted in increased allele recovery and improved intra- and inter-locus peak balance.     

Developmental validation of the AmpFℓSTR® NGM SElect™ PCR Amplification Kit: A next-generation STR multiplex with the SE33 locus

The AmpFℓSTR^® NGM SElect™ PCR Amplification Kit is a new 17-plex STR genotyping kit designed for use primarily in forensic casework analysis. The kit was designed to be a counterpart to the AmpFℓSTR^® NGM™ Kit for laboratories wishing to add the SE33 locus to the new European Standard Set of STR loci. The NGM SElect Kit shares the same primer sets for 16 common loci with the NGM Kit (D10S1248, D3S1358, vWA, D16S539, D2S1338, amelogenin, D8S1179, D21S11, D18S51, D19S433, TH01, FGA, D22S1045, D2S441, D1S1656 and D12S391), with additional primers for the SE33 locus. Developmental validation studies followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines for STR kit manufacturers and tested several critical areas of kit performance including a sensitivity series, DNA mixtures and inhibited samples. The studies demonstrated that the NGM SElect Kit provides equivalent overall performance to the NGM Kit, but with even greater discriminatory power due to the inclusion of the highly informative SE33 locus.     

Development of STR profiles from firearms and fired cartridge cases

Fired cartridge cases are a common type of evidence found at crime scenes. However, due to the high chamber temperatures and touch nature of this evidence, DNA testing is not commonly sought because it is believed DNA is only present in low levels, whether it is due to initial low levels of DNA and/or DNA degradation from the heat or inhibition of the PCR reaction. Moreover, very few laboratories report STR typing success with fired cases. This study focused on obtaining STR profiles from fired cartridge cases using the AmpFℓSTR^® MiniFiler™ kit, which is designed to amplify DNA from low level, inhibited, and degraded samples. Comparisons to other STR amplification kits were also conducted. In attempt to simulate casework, random individuals loaded cartridges into a firearm. DNA was recovered from the fired cartridge cases using the double swab technique and extracted using an automated large volume DNA IQ™ method. Initially, testing focused on known shedders handling cartridges for 30 s prior to firing. A significantly greater number of alleles was obtained following amplification with the MiniFiler™ kit versus the PowerPlex^® 16 BIO kit. No alleles were observed using the Identifiler^® kit. In an attempt to better simulate casework, a random selection of laboratory personnel handled shotshells for as long as needed to load and fire the weapon. In this mock sample study, the MiniFiler™ kit successfully amplified an average of 22% of expected alleles from DNA recovered from shotshell cases versus the PowerPlex^® 16 BIO kit where an average of 7% of alleles were observed. However, the total number of alleles obtained from the two kits was not significantly different. The quality of the DNA obtained from fired cases was studied with evidence of inhibition in at least 11% of shotshell case samples. After swabbing the head and the hull of three shotshell cases separately, a significantly greater number of alleles was obtained from the hull as opposed to the head of the fired shotshell case. In addition, after firing, various internal firearm surfaces were swabbed, including the chamber of barrel, ejection port, and breechface, in an attempt to obtain amplifiable DNA. DNA was obtained from the chamber of the barrel and was amplifiable using the MiniFiler™ kit, although mixtures were obtained with extensive drop-in and drop-out making this analysis unlikely to aid an investigation.     

Performance of two 17 locus forensic identification STR kits-Applied Biosystems's AmpFℓSTR® NGMSElect™ and Promega's PowerPlex® ESI17 kits

We compared the performance of two recently released 17 loci STR multiplexes for human identification: Applied Biosystems's AmpF?STR^® NGMSElect? and Promega's PowerPlex^® ESI17. The comparative parameters were chosen by their relevance in forensic identification and particularly in crime cases. The comparative analyses encompass: amplification ability, heterozygote balance, allelic drop-out, drop-in, stutter analysis and inter-locus balance.Four DNA profiles were analysed in various concentrations in a serial dilution experiment. The amounts of DNA in the PCR ranged from 3 pg to 420 pg and were analysed in triplicate using 28, 29 and 30 PCR cycles. In order to compare the kits, aliquots from each sample were analysed with both kits under identical conditions. Furthermore, DNA profiles from 200 reference profiles were analysed using both kits.The results from the statistical analyses did not indicate any substantial differences of practical relevance between the kits for forensic case work. For all parameters included in this comparative study, the two kits showed no departure from previously observed patterns relative to e.g. the amounts of DNA or amplicon lengths. Based on our analyses, both kits are considered applicable for forensic crime case work.     

Allele frequency data of 15 autosomal STR loci in four major population groups of South Africa

Allele frequency distributions for 15 tetrameric short tandem repeat (STR) loci were determined using the AmpFlSTR® Identifiler Plus? PCR amplification kit. There was little evidence of departures from Hardy–Weinberg equilibrium or association of alleles of different loci in the population samples. The probability of identity values for the different populations range from 1/3.3?×?10¹⁷ (White) to 1/1.88?×?10¹⁸ (Coloured). The combined probability of paternal exclusion for the different population groups ranges from 0.9995858 (Coloured) to 0.9997874 (Indian).