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.     

Long Allele Designations at D2S1338 and D19S433 Loci as Influenced by Various Multiplex STR Kits

European forensic laboratories are replacing the STR multiplex kits with the new generation 16/17 STR kits. This study examines the influence of the new generation kits and the new Applied Biosystems 3500xL Genetic Analyzer on the designation of long D2S1338 and D19S433 off‐ladder alleles. Different allele calls were obtained using the new NGM? (Applied Biosystems) and PowerPlex^® ESI? (Promega) kits compared with AmpF?STR^® SGM Plus? kit (Applied Biosystems). Sequence analysis was used to determine accurate allele designation. The new multiplex kits and the 3500xL Genetic Analyzer improved accuracy of long allele designations. DNA databases worldwide include countless profiles obtained by previous kits. Discrepancies between the new and former technologies may cause failure to detect hits. Discordance is expected due to primer sequence differences between various kits. An additional discordance, occurring in long alleles, independent of primer sequence is reported in this study.     

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.     

Discordance at D3S1358 locus involving SGM Plus™ and the European new generation multiplex kits

During the course of routine database sample analysis in the Israel Police DNA database, an off-ladder D3S1358 allele, calculated to be >22.1, extending into the adjacent vWA locus was observed using Applied Biosystems SGM Plus™ kit.To verify the size of this D3S1358 long allele and to ensure it was not part of a trialle pattern in the neighboring locus, the sample was amplified using three of the European new generation STR multiplex kits: NGM^TM (Applied Biosystem), Powerplex™ ESX and ESI (Promega). The results of these amplifications determined the variant to be a 22 allele. Subsequent sequencing confirmed this designation and revealed a nucleotide polymorphism. Ten additional SGM Plus™ profiled samples with D3S1358 alleles larger than 19, were re-analyzed using NGM^TM and Powerplex™ ESX which also showed discordance in the calculated results between original SGM Plus™ designations and those obtained with the European new generation multiplexes.     

AmpFlSTR MiniFiler™ PCR amplification kit: The new miniSTR multiplex kit

The AmpFlSTR^® MiniFiler™ PCR amplification kit (Applied Biosystems), a new available 8-miniSTR and the sex determining marker Amelogenin multiplex, includes the most common problematic loci (above 200 bp) of the AmpFlSTR^® Identifiler™ PCR amplification kit: FGA, D21S11, D18S51, D13S317, D7S820, D16S539, CSF1PO and D2S1338.Several casework samples with different DNA contents were tested.Results allowed to complete partial Identifiler™ profiles and additional information was achieved in low copy number (LCN) samples, revealing that this miniSTR kit can improve identification of compromised samples.     

Characterization of the variant allele 9.2 of Penta D locus

DNA profiles of forensic cases of Córdoba Province, Argentina, typed by PowerPlex 16 kit (Promega), have shown in the Penta D locus few samples with a variant allele migrating as an off ladder between alleles 9 and 10. In order to determine the molecular basis of the new variant allele, three samples were subject to polymerase chain reaction amplification of the Penta D locus by monoplex, and were further purified and sequenced. The sequence analysis revealed that the off ladder allele has ten repeats motifs AAAGA as allele 10, with three nucleotides (TAA) deletion in the 3' flanking region, 128 nucleotides after the last repeat. Therefore, the variant allele could be explained by a deletion of allele 10, and was designated 9.2. Mse I digestion assay allows to corroborate allele 9.2 without sequencing. A population study in Córdoba Province indicates that allele 9.2 of Penta D locus has a frequency of 0.0063.     

The Use of Hemastix® and the Subsequent Lack of DNA Recovery Using the Promega DNA IQTM System

Abstract:  Following implementation of our automated process incorporating the Promega DNA IQ^TM system as a DNA extraction method, a large number of blood-containing exhibits failed to produce DNA. These exhibits had been tested with the Hemastix^® reagent strip, commonly used by police investigators and forensic laboratories as a screening test for blood. Some exhibits were even tainted green following transfer of the presumptive test reagents onto the samples. A series of experiments were carried out to examine the effect of the Hemastix^® chemistries on the DNA IQ^TM system. Our results indicate that one or more chemicals imbedded in the Hemastix^® reagent strip severely reduce the ability to recover DNA from any suspected stain using the DNA IQ^TM magnetic bead technology. The 3,3',5,5'-tetramethylbenzidine (TMB) used as the reporting dye appears to interact with the magnetic beads to prevent DNA recovery. Hydrogen peroxide does not seem to be involved. The Hemastix^® chemistries do not interfere in any way with DNA extraction performed using phenol-chloroform. The incompatibility of the Hemastix^® chemistries on the DNA IQ^TM system forced us to adopt an indirect approach using filter paper to carry out the presumptive test.     

Primer binding site mutations affecting the typing of STR loci contained within the AMPFlSTR SGM Plus kit

The Forensic Science Service carries out human identification and familial investigations using the AMPFlSTR SGM Plus kit (PE Biosystems, Warrington, England). We have studied approximately 42,000 parent/child allelic transfers (meioses) for deviations from expected Mendelian Inheritance patterns. Of 55 apparent mutations detected, 20 had patterns suggestive of the presence of a primer binding site mutation producing a silent/null allele. The presence of a silent allele was unequivocally demonstrated in 13 of the 20 suspected cases by using alternative primer sets. Of the 13 confirmed cases, 9 involved the D18S51 locus. As the individuals in these cases all originated from the same geographic region of the Middle East, this cluster suggests the presence of a relatively common variant D18S51 allele in that particular group. These data taken together with our previously published work, confirm that the primer binding sites utilised for amplification of the loci contained in the AMPFlSTR SGM Plus kit have highly conserved nucleotide sequences.     

STR data for the AmPFlSTR SGM plus from a regional population of Austria.

Allele frequencies for 10 microsatellite loci--D3S1358, vWA, D16S539, D2S1338, D19S433, THO1, FGA, D8S1179, D21S11 and D18S51--were determined in an Austrian Caucasian population sample from Vienna using the AmpFlSTR SGM plus amplification kit (Applied Biosystems). This study was done on a population sample of 609 unrelated individuals from the city of Vienna.     

Population genetic studies on the tetrameric short tandem repeat loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820 in Egypt.

The short tandem repeat loci (STRs) D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820 and a locus allowing for sex-discrimination (amelogenin) can be co-amplified by the polymerase chain reaction using a commercially available kit (AmpFlSTR Profiler plus, Perkin-Elmer Biosystems, San Jose, CA) and subsequently typed using capillary electrophoresis (ABI Prism 310 Genetic analyzer, Perkin Elmer Applied Biosystems, San Jose CA). To establish databases for these loci for an Arab population sample from Egypt, 140 unrelated persons were typed. Analysis of these data revealed that all loci except for VWA were in Hardy-Weinberg equilibrium, that the combined mean paternity exclusion chance (MEC) was 0.999875 and that the combined discriminating power (DP) was 2.635 x 10(-11). The allelic distributions found in the Egyptian sample were significantly different at four loci from those found for an Austrian Caucasian population, at all nine loci from an African-American sample and at six of six loci from a Chinese sample. No evidence of linkage equilibrium between any of the co-amplified loci was found. Our results support that the combination of multiplex PCR and capillary electrophoresis can both save time and yield excellent results for paternity testing and stain analysis.     

Population data on the AmpFlSTR SGM plus PCR amplification kit in Germans and Austrians

Allele frequencies for the short tandem repeat loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, THO1 and FGA were determined in 231 German and 100 Austrian unrelated Caucasoids using the AmpFlSTR SGM plus kit (Applied Biosystems). All loci met Hardy-Weinberg expectations. In 212 meioses per locus, one mutation event for D19S433 was observed.     

广东汉族人群D7S809的基因频率及其在亲子鉴定中的应用

利用PCR和聚丙烯酰胺凝胶电泳分型技术,调查了以四核苷酸为重复单位的位点D7S809在广东人群的群体资料。在190个被调查的个体中,共发现14个等位基因和50种基因型。经计算杂合率、个人识别率和非父排除率分别为0.8613、0.9645和0.7184,基因型分布符合Hardy-Weinberg平衡。D7S809位点已成功地应用于100例亲权鉴定案中。D7S809是一个高度多态性、稳定、易于分型的位点,在法医学上极有应用价值。     

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.     

Two examples of null alleles at the D19S433 locus due to the same 4 bp deletion in the presumptive primer binding site of the AmpFlSTR Identifiler kit

Two cases of allelic loss at the D19S433 locus after multiplex PCR with the AmpFlSTR Identifiler kit (Applied Biosystems) are described. In both cases the failure of PCR resulted in genetic inconsistencies due to opposite homozygosity. After singleplex PCR with published primers additional alleles were observed and Mendelian inheritance was restored. These PCR products were sequenced and in both cases the same 4 bp deletion near the 3′ end of the repeat region was detected in two alleles of different length. The frequency of these null alleles (two events in 1026 allelic transfers) amounts to 0.0019 (95% confidence limits: 0.0002-0.0070).     

A Multi-Drug Intoxication Fatality Involving Xyrem® (GHB)

Abstract:  Gamma-hydroxybutyrate (GHB) is best known as a recreational depressant drug, whose use has also been implicated in drug facilitated sexual assault cases. It is also available as a therapeutic agent (Xyrem^®) used for the treatment of daytime sleepiness or cataplexy associated with narcolepsy. This is a report of a case of a 53-year-old woman undergoing treatment with Xyrem^® for narcolepsy. The decedent was also prescribed tramadol, gabapentin, cetirizine, modafinil, carisoprodol, and Xyrem^®. Toxicological analysis of the blood revealed GHB 165.6 mg/L, and 90.7 mg/L in the urine. Blood GHB concentrations in the range 156–260 mg/L have been reported to induce moderately sound sleep. The combined use of central nervous system depressant drugs, together with her problematic sleep apnea, and snoring (both contraindications for GHB use) were determined to have caused this subject's death. The manner of death was determined to be accidental.     

Powerplex® Y 23 System: Molecular characterization of a null allele at locus DYS549

We observed a null allele pattern at locus DYS549 in a male subject from North-East Italy typed with the PowerPlex^® Y 23 System (Promega). To investigate whether this pattern was due to the presence of a microdeletion/mutation in primer binding sites or in the locus target region, the sample was amplified with our designed DYS549 primers obtained from GenBank sequence (GDB: 515022). After amplification, a normal hemizygous genotype at this locus was generated, thus indicating the presence of a point mutation in the binding site of the original primer set of PowerPlex^® Y 23 System (Promega). This was further confirmed by sequence analysis, carried out with the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems), according to the manufacturer's instructions. Sequences were run on the ABI Prism 3130 Genetic Analyzer (Applied Biosystems) and analyzed using the Sequencing Analysis v.5.3.1 and the SeqScape v2.6 softwares (Applied Biosystems). Ascertainment of the frequency of null alleles generated from variations at primer binding sites of short tandem repeats loci is of great importance in forensic genetics.     

Validation of the AMPFℓSTR® MiniFilerTM PCR Amplification Kit for Use in Forensic Casework*

Abstract: The AmpF?STR^® MiniFiler^TM PCR Amplification Kit is designed to genotype degraded and/or inhibited DNA samples when the AmpF?STR^® Identifiler^TM PCR Amplification Kit is incapable of generating a complete genetic profile. Validation experiments, following the SWGDAM guidelines, were designed to evaluate the performance of MiniFiler. Data obtained demonstrated that MiniFiler, when used in conjunction with Identifiler, provided an increased ability to obtain genetic profiles from challenged samples. The optimum template range was found to be between 0.2 and 0.6 ng, with 0.3 ng yielding the best results. Full concordance was achieved between the MiniFiler kit and Identifiler kit except in a single case of a null allele at locus D21S11. Numerous instances of severe heterozygous peak imbalance (<50%) were observed in single source samples amplified within the optimum range of input DNA suggesting that caution be taken when attempting to deduce component genotypes in a mixture.     

D16S539 microvariant or D2S1338 off-ladder allele? A case report about a range overlapping between two loci

All forensic laboratories routinely use commercial kits and softwares for automated typing; in rare cases genotyping misinterpretations or mislabellings occur. This study refers to the investigation on a D2S1338 off-ladder allele mislabelling observed in DNA profile of murdered woman.The Identifiler^® revealed heterozygosity in the range of D16S539, with a presumptive microvariant allele “14.2”, based on assigned size, while PowerPlex^®16 resulted in a homozygosity of allele “11”. Singleplex amplification of D16S539 locus confirmed homozigosity. D2S1338 locus, the closest to D16S1338 in Identifiler^®, genotyped as homozigote “19”, was singleplex amplified. The off-ladder peak was gel-isolated, sequenced and designed as a rare “11” allele variant [(TGCC)6(TTCC)5]. Genotype was finally designed as D16S539 “11,11” and D2S1338 “11,19”.To avoid genotyping misinterpretations or mislabelling, ambiguous genotypes should be established by two commercial kits at least. Furthermore, off ladder alleles as well as allele microvariants should be assigned by direct sequencing. This issue should be considered in Criminal DNA database requirements, that is still under debate in Italy.     

Allele and genotype frequencies for the STR locus D18S51 in a western German population

Genetic marker typing based on DNA amplification by the polymerase chain reaction (PCR) increasingly is being employed in forensic casework and for paternity testing. Allele frequencies were determined using PCR for 102 unrelated Germans (Rhine area) for the locus D18S51. Twelve alleles were observed, with frequencies ranging from 0.005 (allele 11) to 0.191 (allele 14). The observed heterozygosity was 0.867, and the power of discrimination was 0.968. There was no deviation from expectations under Hardy-Weinberg assumptions (P = 0.451).     

Developmental validation of a single-tube amplification of the 13 CODIS STR loci, D2S1338, D19S433, and amelogenin: the AmpFlSTR Identifiler PCR Amplification Kit

Analysis of length polymorphism at short tandem repeat (STR) loci utilizing the polymerase chain reaction (PCR) process has proven to be an ideal assay for human identification purposes. The short length of STR loci coupled with the amplification of target sequence through PCR allows for a robust, sensitive, and specific assay for highly polymorphic markers. A multiplex containing fifteen STR loci plus the gender-determining locus Amelogenin was developed to provide a single amplification/detection of all CODIS (Combined DNA Index System) STR loci (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, TH01, TPOX, and vWA) as well as two internationally-accepted STRs (D2S1338 and D19S433). By incorporating five-dye fragment analysis technology and non-nucleotide linkers, previously optimized AmpFlSTR kit primer sequences have been maintained. This kit has been developed in accordance with the standards of the forensic community as defined by the DNA Advisory Board. Validation studies were performed to include developmental validation, and the results support the use of the AmpFlSTR Identifiler PCR Amplification Kit for human identity and parentage testing.