Validation of a 16-locus fluorescent multiplex system

STR multiplexes have been indispensable for the efficient genotyping of forensic samples. The PowerPlex 16 System contains the coreCODIS loci, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, THOI, TPOX, vWA, the sex determinant locus, amelogenin, and two pentanucleotide STR loci, Penta D and Penta E. This multiplex satisfies the locus requirements for most national databases and is the most efficient currently available system due to its single PCR amplification. To provide the groundwork for judicial acceptance, including the publication of primer sequences, and to evaluate laboratory-to-laboratory variation, a developmental validation for casework on this commercially available system was performed in 24 laboratories and produced the following conclusions. Amplification was reliable on a variety of thermal cyclers and product could be analyzed on either an ABI PRISM 310 Genetic Analyzer or an ABI PRISM 377 DNA Sequencer. Genotyping using single source samples was consistent between 0.25 and 2 ng of input DNA template with a few laboratories obtaining complete genotypes at 0.0625 ng. However, heterozygote allele imbalance (<60% peak height balance) caused by stochastic effects was observed at a rate of 13% with 0.125 ng DNA and 22% at 0.0625 ng DNA. Mixture analyses were done using a total of 1 ng of DNA template. Most alleles were detected in mixtures of 4 to 1 and some minor alleles were detected in mixtures of 19 to 1. Optimum amplification cycle number was dependent on the sensitivity of the detection instrument used and could also be adjusted to accommodate larger amounts of DNA on solid supports such as FTA paper. Reaction conditions including volume, annealing temperature, and concentrations of primer, AmpliTaq Gold, and magnesium were shown to be optimal yet robust enough to withstand moderate variations without affecting genotype analysis. Environmental, matrix and standard source analyses revealed an ability to obtain complete genotypes in all sample types except those exposed to 80 degrees C for 12-48 days. Finally, comparison of genotype results from the PowerPlex 16 System with other commercially available systems on non-probative reference and forensic samples showed consistent results.     

Characterization and validation studies of powerPlex 2.1, a nine-locus short tandem repeat (STR) multiplex system and penta D monoplex

In order to increase the power of discrimination for human identification purposes, a nine-locus short tandem repeat (STR) multiplex, the GenePrint PowerPlex 2.1 system (PowerPlex 2.1) developed by Promega Corporation and a separate pentanucleotide-repeat locus, Penta D, were tested. This megaplex system includes the highly polymorphic loci FGA, TPOX, D8S1179, vWA, Penta E, D18S51, D21S11, TH01, and D3S1358 and may be used in combination with the eight-locus STR multiplex, the GenePrint PowerPlex 1.1 system (PowerPlex 1.1) that has been previously developed. Three of the loci, TPOX, TH01 and vWA, have been included in both systems for quality control purposes. As with PowerPlex 1.1, PowerPlex 2.1 is also based on a two-color detection of fluorescent-labeled DNA products amplified by polymerase chain reaction (PCR) and provides a valuable tool for accurate and rapid allele determination. The primer sequences used in the PowerPlex 2.1/Penta D system are also presented in this report. To meet the "Quality Assurance Standards for Forensic DNA Testing Laboratories" (FBI), we tested the efficiency and reproducibility of the PowerPlex 2.1/PentaD system by several validation studies that were conducted as a joint project among seven laboratories. Validation tests included concordance studies, sensitivity, and species specificity determination, as well as performance in forensic and environmentally impacted samples. The results produced from these tests demonstrated the consistency and reliability of the PowerPlex 2.1/Penta D system.     

Population data for 11 STR loci in northeast China Han

Allele frequencies for the nine tetrameric short tandem repeats (STR) loci D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, and D7S820 (AmpFlSTR Profiler Plus PCR amplification kit, PE Applied Biosystems) and two pentameric STR loci Penta D and Penta E (PowerPlex 16 system, Promega Corporation) were determined in a population sample of unrelated China Han.     

Allele frequencies for STR loci of the Powerplex 16 multiplex system in five endogamous populations of India

Allele frequencies for the 15 STR locus of PowerPlex 16 were analyzed in 95 healthy unrelated individuals belonging to five important population groups inhabiting different part of India. Fifteen loci studied are Penta E, D18S51, D21S11, THO1,D3S1358, FGA, TPOX, D8S1179, vWA, Amelogenin, Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818. In addition of all tetra nucleotide loci, two penta nucleotide loci Penta D and Penta E of the studied system are also found highly polymorphic in all the five studied populations of India. These loci are found highly informative in solving paternity cases and other forensic testing in studied population.     

PowerPlex~(TM) 16体系在中国人群中罕见等位基因及其类型

目的 分析PowerPlexTM 16体系基因座在中国人群中的罕见等位基因及其类型。方法 应用PCR-STR和DNA序列分析技术,对4650个无关个体在15个STR基因座中的罕见等位基因进行检测。结果 在PowerPlexTM16体系中的D7S820、D16S539、Penta E基因座,检测到2种类型的罕见等位基因,而TH01、D21S11、D5S818、D13S317、Penta D、D8S1179、TPOX、FGA基因座检测出1种类型。其等位基因频率均较低(0.215‰-7.097‰)。结论 超出ladder范围的罕见等位基因序列比相邻等位基因增加(或减少)1个或数个重复单位,因碱基的插入或缺失的罕见等位基因出现在两等位基因之间。     

Allele frequencies for 15 short tandem repeat loci in a representative sample of Bosnians and Herzegovinians

Allele frequencies for the 15 STR loci (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, FGA) included in the PowerPlex 16 kit were obtained from a multiethnic sample of 100 unrelated individuals born in Bosnia and Herzegovina.     

Validation of the PowerPlex 1.1 loci for use in human identification

STR typing is now the favored method of DNA analysis for the purposes of human identification in the forensic community. The Forensic Services Division of the Detroit Police Department has completed its validation of the PowerPlex 1.1 loci (CSF1PO, TPOX, THO1, vWA, D16S539, D7S820, D13S317, and D5S818) for use in forensic casework. Detroit Metro Area Red Cross samples were typed from each of five racial/ethnic groups--the Hispanic, Caucasian, African American, Asian, and American Indian populations--and allele and genotype frequencies were calculated. A rare off-ladder variant (9.1 allele at D7S820) was identified among the database samples. A number of validation studies were performed. DNA was extracted from different substrates and typed as expected, except for the DNA extracted from leather (signal absent from the D16S539, D7S820, D13S317, CSF1PO, and TPOX loci) and from dirt (no PCR product generated). The minor contributor in the mixture study (250 pg input DNA) was facile to discern. The Concordance study, the variety of fluids from the same individual, and NIST standards studies all produced the expected results. Finally, STR data confirmed previous DNA typing results from adjudicated casework samples.     

DNATyper^TM15试剂盒的确证试验

目的测试DNATyper^TM15试剂盒的技术性能指标，评估其法医学应用能力。方法制定测试方案，从方法学验证、灵敏度、混合样本、批次间试剂稳定性及批量样本测试、DNA提取方法适应性测试、各类常见检材的测试、稳定性测试等8个方面进行测试。并与Identifiler^TM PowerPlex16剂盒进行比较。结果DNA Typer TM15试剂盒灵敏度较高，批次间性能稳定，对各类案件检材和DNA提取方法具有较好的适应性，具有检验混合DNA样本检测的能力。结论DNA Typer TM15在上述性能指标等方面已经达到国际同类产品的技术水平，可用于法庭科学的检案与建库。     

Allele frequencies for 15 STR loci in Van-Ağri districts of the Eastern Anatolia region of Turkey

Allele frequencies of 13 tetrameric short tandem repeat (STR) loci (D3S1358, TH01, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, D8S1179, TPOX, FGA) and 2 pentameric short tandem repeat loci (Penta E and Penta D) included in the PowerPlex 16 kit were obtained from a sample of 116 unrelated individuals in Van-A?ri districts of the Eastern Anatolia region of Turkey. The expected performance of these loci for personal identification and paternity testing in this population was estimated.     

Development and validation of the AmpFlSTR MiniFiler PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA

DNA typing of degraded DNA samples can be a challenging task when using the current commercially available multiplex short tandem repeat (STR) analysis kits. However, the ability to type degraded DNA specimens improves by redesigning current STR marker amplicons such that smaller sized polymerase chain reaction (PCR) products are generated. In an effort to increase the amount of information derived from these types of DNA samples, the AmpFlSTR MiniFiler PCR Amplification Kit has been developed. The kit contains reagents for the amplification of eight miniSTRs which are the largest sized loci in the AmpFlSTR Identifiler PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA). Five of these STR loci (D16S539, D21S11, D2S1338, D18S51, and FGA) also are some of the largest loci in the AmpFlSTR SGM Plus kit. This informative nine-locus multiplex, which includes the gender-identification locus Amelogenin, has been validated according to the FBI/National Standards and SWGDAM guidelines. Our results demonstrate significant performance improvements in models of DNA degradation, PCR inhibition, and nonprobative samples when compared to the AmpFlSTR Identifiler and SGM Plus kits. These data support that the MiniFiler kit will increase the likelihood of obtaining additional STR information from forensic samples in situations in which standard STR chemistries fail to produce complete profiles.     

Genetic profile of a multi-ethnic population from Guiné-Bissau (west African coast) using the new PowerPlex 16 System kit

Allele and haplotype frequencies of 15 chromosome STR loci included in the kit PowerPlex16 System from Promega, were determined in a sample of unrelated males from Guiné-Bissau, a country from the west African coast. All individuals were subjected to an interview in order to make sure that their ancestors belonged to the same ethnic group. This way we intended to look for possible inter-ethnic differences. PowerPlex 16 includes STRs not studied before in any multi-ethnic population. The kit includes two new allele markers (Penta D and Penta E), which are very useful either in forensics or population genetic studies. The Guinean population presents significant differences when compared with other African populations.     

Distribution of alleles of 15 STR loci of the Powerplex 16 Multiplex system in four predominant population groups of South India

Allele distribution of the 15 STR loci of Powerplex 16 Multiplex System were studied in four predominant population groups of South India for evaluating their significance in human identification and population study: Iyengar Brahmin (65), Gowda (59), Lingayat (98) and Muslim (45) from the state of Karnataka. The loci analyzed are--D3S1358, THOI, D2ISI I, D18S51, D5S818, Penta E, D13S317, D7S820, D16S539, CSFIPO, Penta D, vWA, D8S179, TPOX and FGA. Out of 15 STR loci Penta D and D18S51 were found highly polymorphic in the studied populations.     

Concordance study between the AmpFlSTR MiniFiler PCR amplification kit and conventional STR typing kits

The AmpFlSTR MiniFiler polymerase chain reaction amplification kit developed by Applied Biosystems enables size reduction on eight of the larger STR loci amplified in the Identifiler kit, which will aid recovery of information from highly degraded DNA samples. The MiniFiler Kit amplifies CSF1PO, FGA, D2S1338, D7S820, D13S317, D16S539, D18S51, and D21S11 as well as the sex-typing locus amelogenin. A total of 1308 samples were evaluated with both the MiniFiler and Identifiler STR kits: 449 African American, 445 Caucasian, 207 Hispanic, and 207 Asian individuals. Full concordance between Identifiler and MiniFiler Kits was observed in 99.7% (10,437 out of 10,464) STR allele calls compared. The 27 differences seen are listed in Table 1 and encompass the loci D13S317 (n = 14) and D16S539 (n = 10) as well as D18S51 (n = 1), D7S820 (n = 1), and CSF1PO (n = 1). Genotyping discrepancies between the Identifiler and MiniFiler kits were confirmed by reamplification of the samples and further testing using the PowerPlex 16 kit in many cases. DNA sequence analysis was also performed in order to understand the nature of the genetic variations causing the allele dropout or apparent repeat unit shift.     

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.     

TWGDAM validation of a nine-locus and a four-locus fluorescent STR multiplex system

The Gene Print PowerPlex 1.1/Amelogenin and FFFL Fluorescent STR Systems have been validated following the recommendations presented by the Technical Working Group on DNA Analysis Methods (TWGDAM). The PowerPlex 1.1/Amelogenin System supports simultaneous amplification of eight short tandem repeat loci and the Amelogenin gender identification marker. The loci D16S539, D7S820, D13S317, and D5S818 are labeled with fluorescein (FL) while the loci CSF1PO, TP0X, TH01, vWA and Amelogenin are labeled with carboxy-tetramethylrhodamine (TMR). The FFFL Multiplex System is composed of the loci F13A01, FESFPS, F13B, and LPL, each labeled with fluorescein. We have observed no overlap of alleles across loci labeled with an individual fluorescent dye. Samples of each system were amplified and labeled in a single reaction, separated by electrophoresis through a denaturing polyacrylamide gel, and amplified alleles detected using a Hitachi FMBIO Fluorescent Scanner. Alterations from the standard amplification protocols in cycle number and annealing temperature generally produced excellent results. In experiments testing sensitivity as little as 0.2 ng of DNA template could be detected. As expected, different body fluids from the same individuals generated identical DNA profile results. Template DNA derived from blood-strains deposited on a variety of matrix supports displayed robust amplification except for material derived from deposits on wood and Japanese orchid leaves. Mixtures of DNA templates could be interpreted with the minor component present in as little as ten percent of the total sample. Monoplex and multiplex amplifications produced identical amplified allele patterns, indicating that STR multiplex systems save template and increase efficiency in the amplification procedure without loss of quality. Analyses of genotype frequencies in African-American, Caucasian-American and Hispanic-American populations using all twelve loci were used to determine matching probabilities smaller than 1 in 1.14 x 10(8) and 1 in 2658 for the PowerPlex 1.1 and the FFFL Multiplex Systems, respectively. The matching probability achieved with the two systems combined is smaller than 1 in 3.03 x 10(11). The independence of alleles within loci was generally demonstrated by applying the exact test to demonstrate Hardy-Weinberg Equilibrium. All of the studies performed indicate that the PowerPlex 1.1/Amelogenin and FFFL Multiplex Systems are powerful, robust, and reliable investigative tools that can be used in the analysis of forensic samples.     

STR data for SGM Plus and penta E and D loci in a population sample from south Poland

Frequency data of short tandem repeats loci included in the SGM Plus kit and on two pentanucleotide STR loci: Penta E and Penta D [Profiles DNA 2 (1998) 2] included in the PowerPlex16 kit were collected from a sample of 400 (for SGM Plus) and 91 (for Penta E and Penta D) random, unrelated individuals born in the South Poland region.     

Concordance study on population database samples using the PowerPlex 16 kit and AmpFlSTR Profiler Plus kit and AmpFlSTR COfiler kit

Over 500 population database samples comprising African Americans, Bahamians, and Southwestern Hispanics were typed using the PowerPlex 16 and the Profiler Plus COfiler kits. There was only one sample in which a typing difference was observed. An FGA heterozygote profile was observed using the PowerPlex 16 primers, and a single allele FGA profile was observed using Profiler Plus primers. Thus, the extant data suggest that the primers used in the PowerPlex 16, Profiler Plus, and COfiler kits are reliable for typing reference samples destined for use in CODIS. In addition, African American, Bahamian, and Southwestern Hispanic databases have been established for the STR loci Penta D and Penta E. Both loci are highly polymorphic. The application of the product rule is valid for estimating the rarity of a multiple loci profile consisting of these two and the 13 core STR loci.     

Allele frequencies of sixteen STRs in the population of Northern Portugal

Allele frequencies of sixteen autossomal short tandem repeats (STRs), D3S1358, VWA, D16S539, D8S1179, D21S11, D18S51, TH01, FGA, D5S818, D13S317, D7S820, TPOX, CSF1PO, Penta D, Penta E (included in the PowerPlex 16 kit), and the SE33 (PowerPlex ES Monoplex System SE33) were determined in a sample of 200 healthy unrelated individuals from the north of Portugal.     

Forensic genetic analysis of the population of Gujarat with PowerPlex 21 multiplex system

The present investigation assessed the allele frequencies and forensic parameters for the loci included in PowerPlex 21 multiplex system (Promega, USA) in 101 unrelated individuals residing in the state of Gujarat, India. The study likewise introduces the first global report on polymorphism on Penta D and Penta E autosomal STR loci from the population of Gujarat, India. The obtained outcomes in the study revealed that the considered STR multiplex system is highly polymorphic and suitable for forensic genetic analysis.     

Genetic diversity at two pentanucleotide STR and thirteen tetranucleotide STR loci by multiplex PCR in four predominant population groups of central India

Genetic diversity study at STR loci in 208 individuals belonging to two backward groups, one caste and one tribal community of Central India called "Chhattisgarh" has been carried out to evaluate significance of Powerplex System loci in human identification and population diversity. Populations are Agharia (72), Satmani (50), Dheria Gond (36) and Teli (50). Fifteen loci (Powerplex 16 Kit) studied are Penta E, D18S51, D21S11, THO1, D3S1358, FGA, TPOX, D8S1179, vWA, Amelogenin, Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818. The studied penta nucleotide STR (two) and 13 tetranucleotide (CODIS ) STR are found to be highly polymorphic genetic markers in all studied populations. Most common allele for the four studied population has been found to be same at THO1 (allele 9), D8S1179 (allele 14), CSF1PO (allele 12), Penta E (allele 11) and D16S539 (allele 11). Penta E is found to be most polymorphic (PD=0.89373) among studied 15 STR loci in four populations of Central India.