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.     

Y-SNPs analysis using two different approaches in a Northern Portugal male population sample

The non-recombining portion of the human Y (NRY) chromosome has various types of variation, including single nucleotide polymorphism (SNP). In spite of their low discrimination power, they provide a powerful and simple exclusion tool for forensic purposes. A special advantage of SNPs is that it can potentially detect smaller DNA fragments (analysis of degraded DNA).The aim of this work consisted in the analysis of a group of SNP polymorphisms (M2, M9, M35, M89, M45, M170, M172, M173, M207 and P25) in a Northern Portugal male population sample, which allows the determination of the most common European haplogroups, including the Northern Portugal ones.The method used for typing these polymorphisms was the real-time PCR with TaqMan probes on the ABI 7000 platform (Applied Biosystems).We had some difficulties in typing some of the markers using this approach. However, the preliminary results obtained for the defined haplogroups are in accordance with those described in close European populations. To confirm the typing and solve the doubts that emerged from the real-time approach, the samples were also typed using SNapShot.     

Developmental validation of the AmpF?STR SEfiler Plus™ PCR amplification kit: An improved multiplex with enhanced performance for inhibited samples

Since its introduction in 2002, the AmpF?STR^® SEfiler™ kit has provided a highly discriminating DNA profiling option to German forensic laboratories by combining the widely used SGM Plus^® Kit loci with the SE-33 locus required for the German DNA Database. Whilst proving successful on database samples, laboratories using the SEfiler™ kit have reported the need for chemistry better able to handle the ever-increasing number of casework samples.The new AmpF?STR^® SEfiler Plus™ kit contains the same loci and primer sequences as the SEfiler™ kit but uses improved synthesis and purification processes to minimize the presence of dye-labeled artifacts. Other improvements include modified PCR cycling conditions for enhanced sensitivity and a new buffer formulation that improves performance with inhibited samples when compared to the original SEfiler™ kit.Validation studies demonstrating the effectiveness of the multiplex are presented with emphasis on the models of inhibition and casework samples.     

Effect of blood stained soils and time period on DNA and allele drop out using Promega 16 Powerplex kit

Blood stained soils may be of great interest in forensic incidents. Amplification of DNA from soil is often inhibited by co-purified contaminants. Different soils types from Pakistan and Turkey were stained with blood and samples were collected systematically after specified intervals. Rapid, inexpensive, large-scale DNA extraction method involving minimal purification was developed. DNA was quantized using Spectrophotometer and Fluorometer and was confirmed by Agarose Gel Electrophoresis. DNA extracted from different soils in different periods showed a remarkable decrease in yield as well as degradation in every extraction. PCR amplification was performed using various DNA targets present in Promega 16 Powerplex^® System kit. Amplification could not carry out in all loci especially in degraded samples taken after 20 days. Allele n locus drop out was noticed which shows that DNA was degraded. For some loci more than 2 alleles were also noticed showing contamination while working with the blood stained soils.     

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 comparison of three automated DNA purification methods in Forensic casework

Manual Chelex^®-100 and organic extractions (phenol/chloroform) are used as routine methods at the Swedish National Laboratory of Forensic Science, SKL. The aim of this study was to find an automated DNA purification system to replace the organic method. The following methods were evaluated and compared to each other and to the organic method used routinely; BioRobot^® EZ1 with EZ1 DNA Investigator Kit and Card (Qiagen), iPrep™ Purification Instrument with iPrep™ ChargeSwitch^® Forensic Kit and Card (Invitrogen), Magnatrix™ 1200 Workstation with the Magnatrix™ gDNA Blood Kit Forensic and two different protocols; Forensic protocol A and B (Magnetic Biosolutions). Blood on fats, cotton swabs, moist snuff, paper towels and leather, post-mortem blood and muscle tissue were extracted with the different methods. DNA concentration and quality of the electropherograms were examined. Individual comparisons between the four extraction methods showed that iPrep™ and Magnatrix™ 1200 gave significantly lower mean quantities compared to BioRobot^® EZ1 and the organic extraction method (p < 0.05). There were no significant differences between the latter two. BioRobot^® EZ1 generated the best results and is in the process of being validated for routine analysis at SKL.     

Forensic applications of stable isotope analysis: Case studies of the origins of water in mislabeled beer and contaminated diesel fuel

This paper describes the use of oxygen (¹⁸O) isotope analysis of water contained in two different materials — beer and diesel fuel — involved in the resolution of two separate cases. In the first case study, it was possible to demonstrate that a sample of beer labelled as premium brand in fact belonged to a cheap brand. The second case related to the contamination of diesel fuel from a service station. The diesel fuel contained visible amounts of water, which caused vehicles that had been filled up with it to become defective. For insurance purposes, it was necessary to determine the source of water. The δ¹⁸O values for the water of nearly all samples of diesel was close to the δ¹⁸O of local tap water at the filling station.     

Allele frequency distribution for 21 autosomal STR loci in Bhutan

We studied the allele frequency distribution of 21 autosomal STR loci contained in the AmpFlSTR^® Identifiler™ (Applied Biosystems), the Powerplex^®16 (Promega) and the FFFL (Promega) multiplex PCR kits among 936 individuals from the Royal Kingdom of Bhutan. As such these are the first published autosomal DNA results from this country.     

Validation of the AmpF?STR SEfiler Plus™ PCR Amplification kit for forensic STR analysis

Validation of the AmpF?STR^® SEfiler Plus™ PCR Amplification kit with 29 and 30 PCR cycles for forensic STR analysis demonstrated that the kit had fewer artefacts than the AmpF?STR^® SGM Plus™ kit (28 PCR cycles). The SEfiler Plus kit was more sensitive and devoid of colour artefacts, but showed more stutters, drop-ins, drop-outs and allelic imbalances.     

An analysis of single and multi-copy methods for DNA quantitation by real-time polymerase chain reaction

The goal of this paper was to examine and compare two different commercially available approaches to the determination of the relative quantities of autosomal and Y chromosomal DNA using real-time PCR. One, Quantifiler^® Duo, utilizes a TaqMan^® assay with single copy probes for both autosomal human and Y quantification. The other method, Plexor HY^® utilizes a primer quenching assay with multi-copy probes for its quantification of autosomal human and Y chromosomal DNA. To test these approaches we have utilized the NIST Human DNA Quantitation Standard Reference Material 2372, a set of three different NIST human DNA quantification standards, to examine the precision, accuracy and sensitivity of the real-time PCR assays. We also examined data from both systems utilizing casework samples. The results show that both systems produced linear estimates for DNA quantity over a broad range of input DNA. However we did observe some apparent copy number effects when comparing the three different NIST standards which we attributed to issues with sequence variations in the different standards. Overall, the single copy approach provided better accuracy while the multi-copy approach produced better sensitivity. Thus the choice of which system to use should depend upon the goals of the user.     

The Upper Silesia (Poland) population and forensic usefulness of 15 autosomal STR loci

Allele frequencies, forensic parameters for the 15 STR loci in the AmpFlSTR^® Identifiler Kit (Applied Biosystems), D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D19S433, VWA, TPOX, D18S51, D5S818 and FGA were determined in a sample of 150 unrelated dead and alive adults from the Upper Silesia region (Poland). The values of heterozygosity (Ht), polymorphic information content (PIC), power of discrimination (PD), matching probability (PM), mean exclusion chance (MEC) and mean exclusion probability (MEP) were calculated. Possible divergence from HWE was determined. Comparison of allele frequencies for examined STR loci between the Upper Silesia population and other Polish populations was carried out.     

Divergent genetic strata in five Bahamian islands

Based on historical records, the genetic landscape of the Bahamian archipelago is presumed to be complex and to exhibit island-specific characteristics, yet the genetic composition of the island chain, which could corroborate or refute these past accounts, remains poorly defined. As such, the current investigation was undertaken to genetically characterize 5 Bahamian populations representing the Northwest (Grand Bahama and Abaco) and Central (Eleuthera, Exuma and Long Island) Bahamas across the 15 autosomal Identifiler loci routinely employed in forensic analyses. Altogether, our findings suggest that Bahamians are a genetically heterogeneous group, with each island sampled receiving differential contributions from African, European, East Asian and Native American sources. Even though the strongest genetic signal in all 5 collections emanates from continental Africa, inter-island differentiation is noted in both the Structure and admixture analyses. The presence of alleles not in common among the 5 insular populations also signals genetic heterogeneity among the islands of the archipelago. This is especially the case when considering the Long Island population, which exhibits statistically significant genetic differences in relation to the other Bahamian collections and the New World groups of African descent (Afro-American and Afro-Caribbean) in the G-test pair-wise comparisons, even after application of the Bonferroni adjustment.     

Validation studies of the ParaDNA® Intelligence System with artificial evidence items

Short tandem repeat (STR) profiling is one of the mostly used systems for forensic applications. In certain circumstances, STR profiling is time-consuming and costly, which potentially leads to delays in criminal investigations. LGC (Laboratory of the Government Chemist, UK) Forensics has developed a robust STR profiling platform called the ParaDNA^® Intelligence Test System which can provide early tactical intelligence and aid investigators in making informed decisions on sample prioritization for detection. Here, we validated the ParaDNA intelligence test for its application in forensic cases using a range of mock evidence items following guidelines set by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Specifically, we tested the sensitivity and accuracy of the ParaDNA intelligence test, as well as the success rates for detecting mock samples and for use in case scenarios. Our findings demonstrate that the ParaDNA intelligence test generates useful DNA profiles, especially for samples such as blood, saliva, and semen that contain ample DNA, indicating the benefits of including ParaDNA as a prior step in forensic STR profiling pipelines.     

The transfer of human DNA by Lucilia cuprina (Meigen) (Diptera: Calliphoridae)

Blowflies leave deposits, termed artefacts, through the processes of excretion and regurgitation. To date, little consideration has been given to the possibility of adult blowflies consuming biological material and subsequently acting as vectors of human DNA through these artefacts. In this study, Lucilia cuprina (Meigen) (Diptera: Calliphoridae) were fed either human blood or human semen ad libitum and their artefacts were analysed for human DNA content. Samples containing 1, 10, 30 and 50 artefacts were tested. Quantifiable and typeable levels of human DNA were found in samples derived from both food sources, and even in samples containing a single artefact. Semen-derived artefacts contained significantly more human DNA than artefacts produced after a blood meal. Consequently a smaller number of artefacts was required to collect sufficient DNA for genotyping. These findings are forensically important as it provides investigators with another potential source of DNA at a crime scene where a body has been moved, or an attempt has been made to clean up biological material. They also highlight how fly artefacts could potentially contaminate and compromise evidence.     

Bhartṛhari and Maṇḍana on Avidyā

The concept of avidyā is one of the central categories in the Advaita of Śaṇkara and Maṇḍana. Shifting the focus from māyā, interpreted either as illusion or as the divine power, this concept brings ignorance to the forefront in describing duality and bondage. Although all Advaitins accept avidyā as a category, its scope and nature is interpreted in multiple ways. Key elements in Maṇḍana’s philosophy include the plurality of avidyā, individual selves as its substrate and the Brahman as its field (viṣaya), and the distinction in avidyā between non-apprehension and misapprehension. A closer investigation shows that Maṇḍana is directly influenced by Bhartṛhari’s linguistic non-dualism in developing the concept of avidyā. This study also compares other key constituents such as vivartta and pariṇāma that are relevant to the analysis of avidyā. As the concept of counter-image (pratibimba) emerges as a distinct stream of Advaita subsequent to Maṇḍana, this study also compares the application of pratibimba in the writings of Bhartṛhari and Maṇḍana.     

What similes in Sāṃkhya do: a comparison of the similes in the Sāṃkhya texts in the Mahābhārata, the Sāṃkhyakārikā and the Sāṃkhyasūtra

In Sāṃkhya similes are an important means to communicate basic philosophical teachings. In the texts similes are frequently used, especially in the Sāṃkhya passages in the Mahābhārata, in the Sāṃkhyakārikā and in the Sāṃkhyasūtra. This paper compares the similes in these three texts and analyses changes in the philosophy as revealed in the similes. A comparison of the similes of Sāṃkhya texts produced over more than one thousand years reveals changes in the emphasis in this philosophical system. The purpose of the similes in the Sāṃkhya passages of the Mahābhārata is to produce an intuitive understanding of the separateness of puruṣa and prakṛti. The similes are designed to lead the listener to understand this basic dualism. In the Sāṃkhyakārikā the most difficult issues are the relationship between prakṛti and puruṣa and the idea of prakṛti working for the salvation of puruṣa. One whole chapter of the Sāṃkhyasūtra is devoted to similes.     

Analysis of 11 tetrameric STRs in wild boars for forensic purposes

STR profiling of animal species has a wide range of applications, including forensic identification, wildlife preservation, veterinary public health protection and food safety. We tested the efficacy of a multiplex PCR-based assay including 11 porcine-specific tetrameric STRs in a population sample of wild boars (n = 142) originating from Piedmont (North West Italy). Multiple deviations from Hardy–Weinberg expectations were observed, mostly due to a reduction in observed heterozygosity indicative of a high degree of inbreeding. A value of θ of 0.046 and an inbreeding coefficient of 0.089 were estimated. Combined power of discrimination and probability of exclusion values for the STR panel were 0.9999999999996 and 0.99989. In order to test the suitability of the method for meat traceability purposes, a domestic pig reference sample (n = 412), consisting of commercial lines commonly used in the meat production process, was also typed. A Bayesian cluster analysis carried out using the observed genotypes, showed a percentage of correct subspecies assignment of individual samples of 0.974 for wild boars and 0.991 for pigs, thus demonstrating the usefulness of the multiplex STR-typing system for discrimination purposes.