AmpFlSTR Identifiler Research Articles

Efficiency of DNA extraction from urine test strips

We used six types of urine test strips and cotton swab for comparing the DNA extraction efficiency. DNA extraction was performed with EZ1 Advanced XL, DNA typing with AmpFlSTR Identifiler Plus PCR amplification Kit, and electrophoresis with the Applied Biosystems 3130xl Genetic Analyzer. Combur-Test, Uriflet S, and UroPaper appear suitable for DNA extraction when using urine test strips as presumptive testing reagents for potential blood.

DNA extraction with silica membrane column from teeth left in soil for more than 60 years

We performed DNA extraction from four teeth left in soil for more than 60 years by three methods (AP method, EP method and AEP method). DNA extraction was performed with QIAamp DNA Investigator Kit, DNA typing with AmpFlSTR Identifiler Plus PCR amplification Kit, and electrophoresis with the Applied Biosystems 3130xl Genetic Analyzer. The AEP method is highly efficient in extracting DNA from teeth left in soil for more than 60 years.

An incest case with three biological brothers as alleged fathers: Even 22 autosomal STR loci analysis would not suffice without the mother.

Here, we report an incest paternity case involving three biological brothers as alleged fathers (AFs), their biological sister and her child that was investigated using the Investigator ESSplex Plus, AmpFLSTR Identifiler Plus/Investigator IDplex Plus and PowerPlex 16 kits. Initial duo paternity investigations using 15-loci autosomal short tandem repeat (STR) analyses failed to exclude any of the AFs. Despite the fact that one of the brothers, AF1, had a mismatch with the child at a single locus (D2S1338), the possibility of a single-step mutation could not be ruled out. When the number of autosomal STR loci analysed was increased to 22 without the inclusion of the mother, AF2 and AF3 still could not be excluded, since both of them again had no mismatches with the child. A breakthrough was possible only upon inclusion of the mother so that trio paternity investigations were carried out. This time AF1 and AF2 could be excluded at two loci (D2S1338 and D1S1656) and six loci (vWa, D1S1656, D12S391, FGA, PENTA E and PENTA D), respectively, and AF3 was then the only brother who could not be excluded from paternity. Subsequent statistical analyses suggested that AF3 could be the biological father of the child with a combined paternity index >100 billion and a probability of paternity >99.99999999%. These findings consolidate the fact that complex paternity cases such as those involving incest could benefit more from the inclusion of the mother than simply increasing the number of STR loci analysed.

Genetic identification of degraded and/or inhibited DNA samples

Genetic identification of degraded biological samples can be a challenge as a consequence of the presence of low template DNA and a high content of PCR inhibitors. The aim of this work was to determine the best combination of extraction protocols and amplification kits to obtain the maximum genetic information from biological samples in different fabrics, subjected to distinct environmental conditions. To accomplish this objective, bloodstains on different fabrics were made and forensic samples identical to those found at crime scenes were simulated. These blood samples were simultaneously subjected to different environmental conditions – water well, inside of a house, buried in three types of soil and on the soil surface in a forest, in different seasons during a maximum period of two years. Washes of the bloodstains with detergent and bleach were also carried out. The samples were then extracted, quantified and, according to their quantification, were amplified with different amplification kits. The AmpFℓSTR MiniFiler was efficient in complementing partial genetic profiles from samples in different environmental conditions, when extracted with Chelex and amplified with AmpFℓSTR Identifiler. The use of mini-STR are of major importance, because they allow adding more genetic information with degraded and/or inhibited samples.

Species specificities among primates probed with commercially available fluorescence-based multiplex PCR typing kits.

To assess species specificities among primates of signals from short tandem repeat (STR) loci included in two commercially available kits, mainly the AmpFlSTR Identifiler kit and additionally the GenePrint PowerPlex 16 system, we analyzed 69 DNA samples from 22 nonhuman primate species representing apes, Old World Monkeys (OWMs), New World Monkeys (NWMs), and prosimians. Each prosimian species and the NWM cotton-top tamarin apparently lacked all STR loci probed. Only one peak, the amelogenin-X peak, was evident in samples from all other NWMs, except the owl monkey. In contrast, several loci, including the amelogenin-X peak, was evident in samples from each OWM species. Notably, for each ape sample, the amelogenin peaks were concordant with morphological gender of the individual. Among the primates, especially in apes, the numbers of alleles for STR loci were increasing according to their phylogenetic order: prosimians<NWMs<OWNs<apes, and so among apes: agile gibbons<white handed gibbons<orangutans<gorillas/common chimpanzees/bonobos. The species specificities among primates for a few commercially released multiplex STR kits examined in this study would contribute to forensic examinations.

Evaluation of advanced multiplex short tandem repeat systems in pairwise kinship analysis

The AmpFLSTR Identifiler Kit, comprising 15 autosomal short tandem repeat (STR) loci, is commonly employed in forensic practice for calculating match probabilities and parentage testing. The conventional system exhibits insufficient estimation for kinship analysis such as sibship testing because of shortness of examined loci. This study evaluated the power of the PowerPlex Fusion System, GlobalFiler Kit, and PowerPlex 21 System, which comprise more than 20 autosomal STR loci, to estimate pairwise blood relatedness (i.e., parent-child, full siblings, second-degree relatives, and first cousins). The genotypes of all 24 STR loci in 10,000 putative pedigrees were constructed by simulation. The likelihood ratio for each locus was calculated from joint probabilities for relatives and non-relatives. The combined likelihood ratio was calculated according to the product rule. The addition of STR loci improved separation between relatives and non-relatives. However, these systems were less effectively extended to the inference for first cousins. In conclusion, these advanced systems will be useful in forensic personal identification, especially in the evaluation of full siblings and second-degree relatives. Moreover, the additional loci may give rise to two major issues of more frequent mutational events and several pairs of linked loci on the same chromosome.

An Accelerated Analytical Process fortheDevelopment of STR Profiles for Casework Samples.

Significant efforts are being devoted to the development of methods enabling rapid generation of short tandem repeat (STR) profiles in order to reduce turnaround times for the delivery of human identification results from biological evidence. Some of the proposed solutions are still costly and low throughput. This study describes the optimization of an analytical process enabling the generation of complete STR profiles (single-source or mixed profiles) for human identification in approximately 5 h. This accelerated process uses currently available reagents and standard laboratory equipment. It includes a 30-min lysis step, a 27-min DNA extraction using the Promega Maxwell(®) 16 System, DNA quantification in <1 h using the Qiagen Investigator(®) Quantiplex HYres kit, fast amplification (<26 min) of the loci included in AmpFℓSTR(®) Identifiler(®), and analysis of the profiles on the 3500-series Genetic Analyzer. This combination of fast individual steps produces high-quality profiling results and offers a cost-effective alternative approach to rapid DNA analysis.

Comparison of the effects of two presumptive test reagents on the ability to obtain STR profiles from minute bloodstains

BackgroundBloodstains often constitute the major physical evidence in criminal investi-gations. In many cases, the bloodstains found by the crime scene examiner are minute, possibly because of dissimulation efforts by the perpetrator to eliminate evidence that reveals his identity. In such cases, short tandem repeat (STR) detection procedures must be performed using the same minute bloodstain evidence on which presumptive tests had been performed earlier. In the present study, two of the most often used presumptive test reagents, phenolphthalein and leucomalachite green, were tested to determine their effects on the ability to obtain STR profiles from minute bloodstains. MethodsDried minute bloodstains obtained from 10 donors were treated with phenolphthalein and leucomalachite green. After various times, genomic DNA was extracted from the treated samples using a QIAamp DNA Micro Kit. DNA was quantified with real-time PCR using a Quantifiler Kit. STR loci were amplified using an AmpFLSTR Identifiler Plus Kit, and the amplified products were separated via capillary electrophoresis in a 3130 Genetic Analyzer. ResultsFull DNA profiles were obtained from all minute bloodstain samples treated with phenolph-thalein when extracted after intervals ranging from 1 h to 1 week. In contrast, the DNA in minute bloodstain samples treated with leucomalachite green was severely degraded, especially after relatively long intervals, leading to poor partial DNA profiles. ConclusionPhenolphthalein is recommended as a safe presumptive test reagent for the detection of blood evidence recovered from crime scenes that might subsequently undergo DNA profiling analysis.

White Cell Serology: An Asset for Selective Blood-DNA Extraction from Mixed Stains for STR Analysis

Forensic analysis of mixed DNA samples poses a potential challenge for the investigator. Male-female mixed DNA samples in sexual assault cases are commonly analyzed in forensic laboratory but in case of male-male and female-female mixed DNA sample from multiple perpetrators in sexual assaults (gang rapes) and homicide cases, it is not easy to discriminate the origin of different donors. We present a protocol for mixed blood stain samples involving agglutination of leukocytes with ABO antisera and using the separated leukocytes for DNA profiling. By using this technique, we were able to differentiate the leukocytes of different individuals. DNA profiles were obtained using AmpFlSTR identifiler plus amplification kit.

Genetic diversity between two Igbo men from Owerri senatorial province as determined by autosomal short tandem repeats, Y-chromosomal short tandem repeats and mitochondrial DNA typing methods

Background: Human identification has recently been optimized following the completion of human genome sequence, by using DNA markers that exhibit the highest possible variation. However, in developing countries such as Nigeria, the application of DNA typing for identification of human subjects either for forensic or medical purposes is inadequate due to the absence of national forensic DNA laboratories and lack of the legislative framework. Materials and Methods: In this study, two male subjects of Igbo origin provided their blood, hair and buccal samples for DNA analysis. DNA was extracted, purified, quantified with human quantifiler™ polymerase chain reaction reaction mix. Hyper-variable segment 1 of the D-loop mitochondrial DNA (mtDNA HVS-1) was amplified, purified and fragments sequenced and analyzed with ABI Genetic Analyzer. A multiplex AmpFlSTR Identifiler kit amplified 15 STR plus amelogenin loci of the nuclear DNA and 16 STR of the Y-chromosome. GeneMapper ID software version 3.2 was used for the analysis of autosomal and Y-chromosome AmpFlSTR. Results: Result show that the mtDNA lineage of UMBACK subject belongs to L3e2b while ELAMBIA is assigned to L3f1b1 haplogroup. Based on the allelic frequency database, both subjects displayed uniqueness in random match probability for the autosomal allelic short-tandem repeat (STR). Based upon a mutation rate of 0.003 for Y-DNA STR markers, the two individuals most likely shared a common paternal ancestor 96 generations ago. Both subjects were assigned to E1b1a haplogroup with a 100% probability, which is consistent with the haplogroup associated with Igbo people in Nigeria. Conclusion: As expected, well-established forensic genetic tools comprising of mtDNA, autosomal and Y-chromsomomal STR typing methods were all found to distinguish two selected Nigerian Igbo individuals with a very high power of discrimination.

Biochemical characterization of Molecular Markers for Human Genetic Identification in Paternity testing by DNA profiling

Human Genome is made of 3 billion nucleotide bases Adenine A, Cytosine C, Thymine T and Guanine G. The DNA sequencing infers the order of nucleotides in the genome. The regions of genome having variation in the sequence are crucial for human genetic identification. The molecular markers of 15 different genomic regions of human with repeat nucleotides sequence of 4 bases in the non-coding regions of genome exhibit a high degree of polymorphism. The biochemical characterization of molecular markers elucidates the chromosomal location of the marker, sequence of repeat units and variation among the individuals for human identification in forensic investigation and genetic inheritance. The 15 molecular markers were analysed for human identification in a mother, child and father trio. The STR loci were amplified by PCR using AmpFlSTR Identifiler Plus PCR amplification kit. The amplified markers were separated through capillary electrophoresis on Applied Biosystems Genetic Analyzer ABI 3100 as per the manufactures instructions. The data was analysed by GeneMapper v3.5 software. The statistical analysis was performed using Bayesian mathematics using in house allele frequency data. The paternity index and probability of paternity were 206759811 and 0.999999999. The DNA test conducted on the samples of mother, child and father trio convincingly established the paternity of the child by perfect match of sequence variation short tandem repeats STR genotypes of the genome with the biological parents. The paternity index of molecular markers and probability of paternity in the human identification are very high. The results of examination of 15 molecular markers with their biochemical characterization conclusively establish the genetic identification.

Population data for 22 autosomal STR loci from Estonia.

Allele frequencies and forensically relevant population statistics of 22 short tandem repeat (STR) loci were determined from 303 unrelated Estonian individuals. The samples were amplified with three kits: the AmpFlSTR(®) Identifiler, the PowerPlex(®) ESI 16 and the PowerPlex(®) 16. No significant deviation from Hardy-Weinberg equilibrium was detected, except for locus D22S1045. Investigated loci are very discriminating in Estonian population, with a combined discrimination power of 0.9999999999999999999999999877. Furthermore, a comparison with previously published frequency data from other nearby populations is presented.

Genetic polymorphism of the 26 short tandem repeat loci in the Chinese Hebei Han population using two commercial forensic kits.

We determined the allele frequencies and forensic parameters for the 26 short tandem repeat (STR) autosomal markers in two commercial kits (the Investigator HDplex and AmpFLSTR(®) Identifiler(®) systems) for 183 unrelated individuals from the Han population of the Hebei Province of China. The 26 STRs were all in Hardy-Weinberg equilibrium. No linkage disequilibrium was detected between any pair of loci. The combined power of discrimination and the combined power of exclusion for the 26 STR loci were 1-7.74E-31 and 1-1.21E-11, respectively. Six rare alleles of D10S2325 were identified and named 20, 21, 22, 23, 24, and 31. All the length of the six rare alleles were out of the range of allelic ladder. We calculated the population pairwise genetic distance based on the allele frequencies, using published population data including German, central Polish, south Dutch, northeastern Polish, south Brazilian, Korean, Sichuan Han of China, and Shanghai Han of China. Also we examined the population pairwise genetic distance of loci included in Identifiler system between Hebei Han and other ethnic population of China. These 26 autosomal STR loci could provide highly informative polymorphic data for paternity testing and forensic identification in the Hebei Han population in China. Because they are all in linkage equilibrium, they could be used together to solve deficient kinship cases or cases with mutations.

A Globally used 15 Short Tandem Repeats (STR) Loci in Forensic Human Identification, with their Allele Frequencies and Statistical Values in the Population of Bahrain.

The aim of this study is to estimate the allelic frequencies of Bahraini population using the AmpFlSTR Identifiler® PCR kit which includes 15 universal Short Tandem Repeats (STR) loci. The samples were buccal swabs collected from 302 unrelated individuals from Kingdom of Bahrain. They are selected randomly from Bahraini population in a where to conduct this study. After going through the whole DNA extraction, quantification, Polymerase Chain Reaction (PCR) amplification, Short Tandem Repeats (STR) typing and statistical analysis we got valuable data and results. The most frequent allele types for each locus were: (D8S1179: 13, D21S11: 29, D7S820: 10, CSF1PO: 11, D3S1358: 16, TH01: 7, D13S317: 12, D16S539: 11, D2S1338: 23, D19S433: 13, vWA: 17, TPOX: 8, D18S51: 14, D5S818: 12, FGA: 23). The highest allele frequencies from them were 0.5 for TPOX: 8, 0.3692 for D5S818: 12 and 0.3626 for D16S539: 11. Maximum Homozygosity (H) was 0.3576 shown in TPOX locus. On the other hand, Maximum Heterozygosity (H2) was 0.9106 shown in D2S1338 locus. As expected the most polymorphic loci which had the highest Polymorphic Information Content were the most discriminating ones: D2S1338 (0.9717), D18S51 (0.9668), FGA (0.9640), while the least discriminated locus was TPOX (0.7202), as the discrimination powers (PD) ranged from 0.7202 (TPOX), to 0.9718 (D2S1338). A t-test was applied to the data generated, and therefore it was normally distributed.

Comparative study of STR loci for typing old skeletal remains with modified protocols of AmpFlSTR Identifiler and AmpFlSTR MiniFiler STR Kits

This study highlights a comparative study of short tandem repeat (STR) loci with modified protocols of AmpFlSTR Identifiler and AmpFlSTR MiniFiler STR Kits for typing 27 old skeletal remains collected from 100–1000-year-old mass graves in Pakistan. DNA profiles were obtained from minute quantities of DNA (even from ≤ 10 pg/μL) with modified protocols of these kits, which is a significant achievement in this study. Consensus profiles were produced for each bone sample. A comparison was carried out between Identifiler and Minifiler successfully genotyped STR loci. Full concordance was perceived in 97.33% (146/150) of the compared STR loci, while discordant STR loci were 2.67% (4/150) of the total successfully genotyped STR loci due to either or both allele drop-out or drop-in. Finally, it was observed that the AmpFlSTR MiniFiler kit promoted the recovery of locus/alleles that failed to type with the AmpFlSTR Identifiler kit and more informative DNA profiles were obtained from old skeletal remains with the AmpFlSTR MiniFiler STR kit compared with the AmpFlSTR Identifiler STR kit.

1025-LBP: Identifying source of eye carcinoma biopsy

Patient MS received a keratolimbal allograft from a deceased donor which did not function. He later received another keratolimbal allograft from his brother DS. Patient and brother are HLA identical. MS later developed a carcinoma of the eye. The Transplantation Immunology Department at the University of Cincinnati was asked to extract DNA from the biopsy of the eye carcinoma to determine the tissue source of the carcinoma (ie deceased donor, living donor, or recipient). Our lab received a sample from the biopsy from the eye and we were able to extract DNA from the sample. Note- our lab did not have a sample of the original deceased donor. Qiagen EZ1 DNA Blood 350 uL Kit Qiagen’s Puregene Tissue Kit ABI AmpflSTR Identifiler We used the Qiagen EZ1 DNA blood kits to extract DNA from blood that was received on MS and DS. For the tissue sample, we extracted DNA using Puregene Tissue Kit and obtained DNA with a concentration of 132 ng/ul and a purity of 1.97. We performed STR on recipient MS, donor DS, and on the eye carcinoma biopsy. We were able to identify informative markers for DS. Based on the STR results we concluded that the carcinoma source was NOT donor, DS. We concluded that the origin of the carcinoma source was from the patient and not transplanted from the deceased donor not the living donor.

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.

Analysis of Positive Control STR Experiments Reveals that Results Obtained for FGA, D3S1358, and D13S317 Condition the Success Rate of the Analysis of Routine Reference Samples

About 120,000 reference samples are analyzed each year in the Forensic Laboratory of Lyon. A total of 1640 positive control experiments used to validate and optimize the analytical method in the routine process were submitted to a multivariate exploratory data analysis approach with the aim of better understanding the underlying sources of variability. The peak heights of the 16 genetic markers targeted by the AmpFℓSTR(®) Identifiler(®) STR kit were used as variables of interest. Six different 3130xl genetic analyzers located in the same controlled environment were involved. Two major sources of variability were found: (i) the DNA load of the sample modulates all peak heights in a similar way so that the 16 markers are highly correlated, (ii) the genetic analyzer used with a locus-specific response for peak height and a better sensitivity for the most recently acquired. Three markers (FGA, D3S1358, and D13S317) were found to be of special interest to predict the success rate observed in the routine process.

AmpF&amp;#x2113;STR&lt;sup&gt;&amp;reg;&lt;/sup&gt; Identifiler&lt;sup&gt;&amp;reg;&lt;/sup&gt; Direct Kit による口腔内細胞からの効率的な STR 型検査法の検証

Direct amplification of short tandem repeats (STR) locus from reference sample without DNA extraction, quantification and dilution processes is one of the solutions to achieve a large amount of DNA profiling quickly. Therefore, in this study, AmpFℓSTR Identifiler Direct Kit (IDD) was tested in the laboratory of the National Police Agency. Buccal cells from 88 volunteers self-collected by using the EasiCollect device were transferred on FTA cards. One disk (φ1.2 mm) is punched from each of the 88 FTA cards and automatically distributed to PCR plates by BSD 1000. The optimal PCR cycles were examined using the PCR plates. PCR plate setup, capillary electrophoresis (CE) plate setup and CE were performed by Biomek FXP, Biomek 3000 and 3130xl Genetic Analyzer, respectively. The threshfold of peak detection was set as 150 relative fluorescence units (RFU), and all other conditions of electrophoresis and analysis were used according to the manufacture's recommendations. Number of proper full profiles obtained from 88 samples were 68 (77.3%), 33 (37.6%), 14 (15.9%) and 3 (3.4%) for 25, 26, 27 and 28 cycles PCR, respectively. Therefore, 25 cycles PCR was optimal for our laboratory. The 68 proper full profiles obtained with 25 cycles PCR were used for validation study. Average of peak height and heterozygous peak height ratio of each STR locus were 1000-2000 RFU and 90% or more, respectively. Intra-color balance and inter-locus balance were almost the same as those in previous study with AmpFℓSTR Identifiler Kit (ID), AmpFℓSTR Identifiler Plus Kit (IDPlus) and IDD. When a threshold value of 150 RFU was applied with the “marker-specific stutter ratio”, proper STR typing was possible. Our study indicated that IDD with 25 cycles PCR was applicable to STR typing of single-source buccal cells transferred on FTA card in our laboratory.

Forensic evaluation of the AmpFℓSTR Identifiler kit in nine Mexican native populations from the pre-Columbian Mesoamerican region

Allele frequency distribution and forensic parameters of the AmpFℓSTR Identifiler kit was determined in nine Mexican Amerindian populations based on 1,040 unrelated individuals from the pre-Columbian region known as Mesoamerica. Hardy-Weinberg equilibrium was demonstrated for most of the short tandem repeats (STRs) in all nine populations. The power of discrimination and exclusion were higher than 0.99999 and 0.997942, respectively. In addition, a brief overview of the genetic relatedness and structure (F st = 2.62 %; p = 0.00000) between these populations is presented.