Profiler Plus Research Articles

Angola Population Data with AmpFℓSTR™ Profiler Plus

Abstract Blood stains from 116 unrelated individuals from Angola (West Africa) were stored using bloodstain card and extracted with the standard Chelex method. PCR amplification was performed in a GeneAmp® PCR System 9600, using the AmpFℓSTR® Profiler Plus™ Amplification Kit, according to the manufacturer's recommendations. Electrophoresis of the PCR products was carried out in an ABI Prism™ 377 DNA Sequencer, using 5% denaturing Long Ranger® gels. Sample genotypes were determined with Genotyper® (version 2.0), by comparison with supplied allelic ladders and an internal size standard (GS-500Rox).

Reduced Volume PCR Amplification Reactions Using the AmpFlSTR® Profiler Plus™ Kit

The forensic community continues to seek improvements in DNA typing methods on aspects such as sensitivity and efficacy. Reducing the volume of the AmpFlSTR Profiler Plus reagents offered greater sensitivity and improved the chance of obtaining useful results for samples with very low quantities of DNA and multiple source samples. On the downside, amplifications initiated with less than 0.4 ng of DNA exhibited a twofold increase in the standard deviation of peak ratios. This research suggested a twofold approach to analyzing samples. For samples with greater than 0.25 ng of DNA, a 25 microL reaction is appropriate. Samples that did not demonstrate quantifiable results, or that have less than 0.25 ng, can be amplified by drying the sample directly in the PCR tube and amplifying in a 5 microL reaction. The analyst can expect at least limited results with as little as 0.03 ng of DNA in the 5 microL reaction.

Extraction of Human Nuclear DNA from Feces Samples Using the Qiaamp DNA Stool Mini Kit

The use of a QIAamp DNA Stool Mini Kit (QIAGEN) for extracting human nuclear DNA from feces samples is reported. This method employs a stool lysis buffer and a unique matrix (InhibitEX tablet) to remove PCR inhibitory substances specific to feces samples. DNA extracted from various amounts of stool and from stool samples exposed to different environmental impacts was successfully amplified and typed using the Profiler Plus Amplification Kit and ABI PRISM 310 Genetic Analyser.

STR data for the Amp FlSTR Profiler Plus loci from Murcia region (SE Spain)

Frequency data of the nine STRs included in the Amp FlSTR Profiler Plus Kit were determined in a sample of 114 unrelated individuals from Murcia region (SE Spain).

Recovery of Genomic Dna from Archived PCR Product Mixes for Subsequent Multiplex Amplification and Typing of Additional Loci: Forensic Significance for Older Unsolved Criminal Cases

A method for genomic DNA recovery from different types of PCR product mixes suitable for multiplex amplification and typing using the Profiler Plus STR typing system has been investigated. The application of this method is of significance in cases where the original DNA samples have been exhausted due to repeated typing analyses in an effort to maximize their evidentiary value. Such cases typically involve samples analyzed using the available DNA typing systems of the time which gave a markedly lower power of discrimination, either alone or in combination, compared to that of modern multiplex STR typing systems. It was found that an effective method for recovering genomic DNA from HLA-DQA1 +PM and CTT triplex amplification mixes, suitable for reproducible achievement of the complete Profiler Plus profile, involved the use of Amicon Microcon-100 microconcentrators. Interestingly, this method was not required to achieve the complete nine STR profile using D1S80 amplification mixes.

Genetic variation of the nine Profiler Plus loci in Russians.

This paper presents allele frequency distributions from a representative population sample of the Russian Federation for the nine loci (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820) which constitute the commercially available Profiler Plus PCR amplification kit. DNA samples of 402 Russian individuals from 57 regions of the Russian Federation were amplified in a multiplex reaction with subsequent genotyping using an ABI Prism 377 DNA sequencer. The population data obtained for genotype and allele frequencies conformed to Hardy-Weinberg expectations (HWE).

Determination of the sensitivity and specificity of sibship calculations using AmpF lSTR Profiler Plus.

In circumstances where a known DNA reference sample from the deceased's belongings or biological parents is not available, more complex kinship analyses are possible. The purpose of the work reported here is to determine the sensitivity and specificity of the sibship analysis utilising multiple STR loci. Using all nine Profiler Plus loci, likelihood ratios for biologically-related siblings ranged from slightly less than 1 to over 45,000. When allelic dropout was mimicked, likelihood ratios ranged from less than 1 to over 1,000. Thus, the results of this study have a direct application to forensic laboratories faced with identifications involving sibling comparisons.

Genetic polymorphism at nine microsatellite

Allele frequencies for the nine STR loci of Profiler Plus STR markers were analyzed in 307 healthy unrelated individuals belonging to four predominant highly important endogamous population groups of Ladakh. The studied loci are found highly informative in human identification and understanding history of peopling of India. This is the first report on DNA Markers on any high altitude desert human population group.

DNA profiling: a valuable tool for quality control of sample logistics including occurrences of suspected sample confusion in a blood donation centre.

A molecular method for analysing whole-blood samples should be established for quality control of plasma sample logistics. DNA profiles of retention samples (plasma) were compared to profiles of recent donations (whole blood). DNA extraction, amplification and detection were performed using the Qiagen DNA Blood Mini kit, the AmpFFISTR Profiler Plus Kit and capillary electrophoresis, respectively. Matched pairs of full profiles were obtained for all samples investigated, therefore no deviation from the standardized procedures was detected. Modified extraction and amplification protocols enabled DNA profiling to be used for the quality control of plasma samples. Hence, DNA profiling can be used in the blood bank as a safe and easy method for quality control of sample logistics.

Is the amelogenin gene reliable for gender identification in forensic casework and prenatal diagnosis?

In humans, the amelogenin gene is present on both the X and the Y chromosomes. However, there are size differences in this gene between these chromosomes, which have been utilised for sexing in forensic casework and prenatal diagnosis. Our study using the AmpFl STR Profiler Plus kit, showed a deletion of Y chromosome-specific amelogenin in five Indian males (1.85%). We propose to call them "deleted-amelogenin males" (DAMs), who but for the detection of the presence of other Y-specific markers (e.g. SRY, STR and 50f2) would have been identified as females. Considering the consequences of the result obtained only using the amelogenin marker, we suggest the use of additional Y chromosome markers for unambiguous gender identification.

Allele Frequency Distribution for Nine Fluorescent Based STR Loci in Tamil Population (South India)

Abstract Blood samples were collected from unrelated Tamil-speaking volunteers. DNA was extracted by the salting out procedure (1). DNA typing was by PCR using 2.5 ng target DNA following manufacturer's instructions (AmpFlSTR — Profiler Plus PCR Amplification Kit — Perkin Elmer). Data were analyzed as per the methods already reported (2–4).

Patterns of genetic diversity at the nine forensically approved STR loci in the Indian populations.

Genetic diversity at the nine short tandem repeat (STR) loci, which are universally approved and widely used for forensic investigations, has been studied among nine Indian populations with diverse ethnic, linguistic, and geographic backgrounds. The nine STR loci were profiled on 902 individuals using fluorescent detection methods on an ABI377 System, with the aid of an Amp-F1 Profiler Plus Kit. The studied populations include two upper castes, Brahmin and Kayastha; a tribe, Garo, from West Bengal; a Hindu caste, Meitei, with historical links to Bengal Brahmins; a migrant group of Muslims; three tribal groups, Naga, Kuki and Hmar, from Manipur in northeast India; and a middle-ranking caste, Golla, who are seminomadic herders from Andhra Pradesh. Gene diversity analysis suggests that the average heterozygosity is uniformly high (>0.8) in the studied populations, with the coefficient of gene differentiation at 0.050 +/- 0.0054. Both neighbor-joining (NJ) and unweighted pair group method with arithmetic mean (UPGMA) trees based on DA distances bring out distinct clusters that are consistent with ethnic, linguistic, and/or geographic backgrounds of the populations. The fit of the Harpending and Ward model of regression of average heterozygosity on the gene frequency centroid is found to be good, and the observed outliers are consistent with the population structure and history of the studied populations. Our study suggests that the nine STR loci, used so far mostly for forensic investigations, can be used fruitfully for microevolutionary studies as well, and for reconstructing the phylogenetic history of human populations, at least at the local level.

Twgdam Validation of Ampf_str•: PCR Amplification Kits for Forensic DNA Casework

Laboratory procedures used in short tandem repeat (STR) analysis were subjected to various scenarios that assessed reliability and identified potential limitations. These validation studies were designed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) and the DNA Advisory Board (DAB) (17,18). Various DNA samples were amplified by the polymerase chain reaction (PCR) using AmpFlSTR PCR Amplification Kits (i.e., AmpFlSTR Green I, Profiler, Profiler Plus, and COfiler kits), detected with ABI Prism instrumentation, and analyzed using GeneScan and Genotyper software. Data acquired in these studies reinforced an existing body of knowledge and expertise regarding application and interpretation of STR typing in the forensic science community. Consistent STR genotypes were detected in various body tissues and fluids. Inter-laboratory comparisons produced concordant genotype results. Quantitative interpretational aids for DNA mixtures were characterized. Ability of the typing systems to type potentially compromised samples reliably was evaluated. Nonprobative case evidentiary DNA was successfully amplified, genotyped, and interpreted. Potential limitations or cautionary factors in the interpretation of minimal fluorescence intensity were demonstrated. Differential amplification between loci was observed when PCR was inhibited; preferential amplification typically was not. Single AmpFlSTR locus amplification did not offer consistent benefit over AmpFlSTR multiplexing, even in cases of DNA degradation or PCR inhibition. During rigorous evaluation, AmpFlSTR PCR Amplification Kits reproducibly yielded sensitive and locus-specific results, as required in routine forensic analyses.

STR primer concordance study

Over 1500 population database samples comprising African Americans, Caucasians, Hispanics, Native Americans, Chamorros and Filipinos were typed using the PowerPlex® 16 and the Profiler Plus™/COfiler™ kits. Except for the D8S1179 locus in Chamorros and Filipinos from Guam, there were eight examples in which a typing difference due to allele dropout was observed. At the D8S1179 locus in the population samples from Guam, there were 13 examples of allele dropout observed when using the Profiler Plus kit. The data support that the primers used in the PowerPlex® 16, Profiler Plus™, and COfiler™ kits are reliable for typing reference samples that are for use in CODIS. In addition, allele frequency databases have been established for the STR loci Penta D and Penta E. Both loci are highly polymorphic.

Influence of cyanoacrylate on the efficiency of forensic PCRs

Cyanoacrylate ester (CA) is commonly used by criminalists to detect latent fingerprints on smooth surfaces. We investigated whether this treatment has an influence on a subsequent DNA typing of biological stains, and on the efficiency of three different forensic PCRs (mtDNA, Y-STR determination and the Profiler Plus kit).Using fluorescence labeled primers and an automated detection system, we could show that the presence of CA led to weaker PCR products. Depending on the DNA extraction method the amplification results were significantly weaker compared to untreated controls. To simulate forensic cases we prepared blood and saliva stains on glass slides, extracted the DNA using two different methods and compared the signal intensities of the amplified DNA fragments. Depending on the extraction methods, the presence of CA significantly hampered the amplification of DNA from small stains whereas there was virtually no difference comparing the amplification results of DNA extracted from bigger stains.

STR data for AmpF/STR Profiler Plus loci in south Poland

Frequency data of short tandem repeats (STR) loci included in the AmpF/STR Profiler Plus kit were collected from a sample of 253 random, unrelated individuals born in the south Poland region. All loci met Hardy–Weinberg expectation.

Population data on nine STRs from Cantabria, a mountainous region in northern Spain

Allele frequencies for nine STRs loci included in the AmpFlTR Profiler Plus kit (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) were obtained from a sample of 158 unrelated individuals living in Cantabria, a region in northern Spain.

STR data for the Amp FlSTR Profiler Plus and COfiler loci from the Maghreb (North Africa)

Allele frequencies for 13 STRs (D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, THO1, TPOX, CSF1PO and D16S539) included in the Amp FlSTR ® Profiler Plus™ and COfiler™ kits were determined for a population sample from the Maghreb (Northern Africa).

TWGDAM Validation of the AmpFℓSTR Profiler Plus and AmpF∓STR COfiler STR Multiplex Systems Using Capillary Electrophoresis

Prior to forensic implementation, a profiling system requires validation following the recommendations presented by the Technical Working Group on DNA Analysis Methods (TWGDAM). In this work two such systems, AmpFlSTR Profiler Plus and AmpFfSTR COfiler have been validated according to the guidelines provided by TWGDAM. Profiler Plus and COfiler simultaneously amplify nine and six STR loci respectively; both also amplify a portion of the amelogenin gene. Performance of the two STR multiplex systems under conditions set forth by TWGDAM was robust and reproducible, indicating that these systems are suitable for use in forensic analysis. Additionally, specific sections of the TWGDAM validation guidelines are especially valuable in terms of familiarizing users with particular limitations of the systems prior to taking on casework.

AmpFℓSTR® Profiler Plus™ and AmpFℓSTR® COfiler™ Analysis of Tissues Stored in GenoFix™, a New Tissue Preservation Solution for Mass Disaster DNA Identification

A preliminary study was conducted to assess the capability of a new alcohol-based tissue fixative, GenoFix, to preserve DNA from biopsy tissues stored at room temperature and/or -20 degrees C in a freezer, for subsequent short tandem repeat (STR) DNA typing analysis. Fresh human smooth muscle samples were stored at room temperature in GenoFix for one month and up to one year and seven months before being processed using the megaplex STR systems, AmpFlSTR Profiler Plus and AmpFlSTR COfiler. Alternatively, muscle tissues in GenoFix were placed at -20 degrees C in a freezer for up to 3 1/2 years following two to three months in the fixative at room temperature. DNA analysis was also carried out on tissues stored in GenoFix for one month at room temperature and subsequently paraffin-embedded and stored at room temperature for four years. The AmpFlSTR Profiler Plus and AmpFlSTR COfiler STR profiles produced, using DNA extracted from all fixed tissue samples, were of very good quality. The fluorescent signals were well balanced across the nine STR loci or six loci comprised in the megaplexes surveyed and profiles showed no differences with those observed for the control blood of the respective donor patients. Continuous exposure to GenoFix at room temperature (up to one year and seven months) did not compromise the STR typing analysis of the fixed tissues. No adverse effects were noted on the STR typeability of tissues fixed with GenoFix and stored at -20 degrees C in a freezer for up to 3 1/2 years. STR profiles generated from the paraffin-embedded tissues fixed in GenoFix were of excellent quality. This preliminary study suggests that GenoFix can be used to store tissue samples at room temperature for up to one year and seven months or at -20 degrees C in a freezer for longer storage (up to 3 1/2 years). This new and odorless tissue fixative promotes tissue and DNA preservation in a very effective manner and as such may prove useful in criminal investigations or mass disaster identifications carried out in remote locations and in which a small or large number of tissue samples are collected for further analyses.