Identification of a D8S1179 primer binding site mutation and the validation of a primer designed to recover null alleles

A population study of Chamorros and Filipinos using short tandem repeat (STR) loci amplified with the AmpFlSTR Profiler Plus PCR amplification kit demonstrated an excess of observed homozygosity at the D8S1179 locus. Use of a different set of D8S1179 primers to type the same samples did not demonstrate an excess of homozygosity and showed discordant genotypes at the D8S1179 locus. A single point mutation, G-to-A transition, 16 nucleotides from the 3' end of the reverse primer, was identified to cause allele dropout when using the AmpFlSTR Profiler Plus primer set. An additional D8S1179 reverse primer specific for the variant was constructed resulting in the recovery of the null allele. The primer was included in the newly developed AmpFlSTR Identifiler PCR amplification kit. No deleterious effects or non-specific peaks were observed in validation experiments evaluating primer concentration, Mg2+ concentration, annealing temperature and population samples.     

False homozygosities at various loci revealed by discrepancies between commercial kits: implications for genetic databases

Routine control of 2055 consecutive genotypes revealed discrepancies between the profiles established with the SGM plus and/or Profiler plus kits on one hand, and the profiles established with the Powerplex16 kit on the other hand. Furthermore, five discrepancies for vWA, three for D8S1179, two for FGA and three for D18S51 loci were found. In 10 cases (loci vWA, FGA, D18S51, D8S1179), the SGM plus and/or Profiler plus profiles showed homozygosity and the Powerplex16 genotype revealed heterozygosities which were confirmed to be true, both by typing with individual primer pairs and DNA sequencing. In four cases (two discrepancies at locus FGA, one at D18S51 and an abnormal paternity pattern for D5S818), the Powerplex16 kit showed apparent homozygosity and the SGM plus and/or Profiler plus kits showed heterozygosity. Mutation analysis could be performed for some of these individuals and evidenced variants, presumably leading to an annealing failure of one primer; the identified mutations are reported. It is suggested that databases should include information about the kits used to determine the profiles while ensuring that the primer sequences are made available.     

Development of two new Mini-STR multiplex assay for typing archival Bouin's fluid-fixed paraffin-embedded tissues

Short amplicon autosomal short tandem repeat (Mini-STR) assay has proved to be a highly useful tool in forensic applications, especially for highly degraded DNA samples that typically result in partial profiles and total loss of information from regular STR amplicons.In this study two new quadruplex systems were designed to get nuclear DNA profile from degraded forensic casework samples. In order to obtain PCR products less than 120 bp in size, primer pairs of eight STR markers, included in available commercially multiplex PCR kits, were redesigned and assembled in two PCR-multiplexes: D8S1179, D3S1358, TPOX, D16S539 and CSF1P0, TH01, D13S317, D5S818.After validation, these two Mini-STR quadruplex were employed in paternity testing case that involved DNA extraction from archival postmortem Bouin's fluid-fixed paraffin-embedded tissue where commercial kit yielded low success. The results obtained with the present Mini-STR PCR-multiplexes proved clearly demonstrating their usefulness in analyzing degraded DNA samples.     

D8S1179基因座等位基因丢失现象的研究

目的 探讨在进行STR分型时发生的等位基因“丢失”情况的原因。方法 分别采用Profiler Plus试剂盒和GenBank数据库设计的引物,对D8S1179基因座进行基因分型,并对丢失的等位基因进行测序分析。结果D8S1179基因座丢失的等位基因在第147位碱基出现G-A转换。在中国人群中,该位置出现碱基转换的频率是0.50×10-2(在2013个无关个体中观察到10例无关变异事件)。结论 第147位碱基转换正好位于Prfiler Plus试剂盒中D8S1179基因座的引物结合区域,导致扩增时等位基因丢失。     

Co-amplification of ENFSI-loci D3S1358, D8S1179 and D18S51: validation of new primer sequences and allelic distribution among 2874 individuals

The present communication presents a new triplex PCR co-amplifying three loci (D3S1358, D8S1179 and D18S51) recommended for STR typing by the European Network of Forensic Science Institutes (ENFSI). Twenty-two different primers were tested to optimise the PCR. Four of the six primer sequences finally chosen were self selected, the fifth was a published one and the sixth derived from a commercially available multiplex kit. Using this PCR-setup, even minimum amounts of genomic DNA are sufficient to analyse the STR loci D3S1358, D8S1179 and D18S51 in parallel. Especially in forensic casework, where DNA is mostly limited and often contaminated with enzyme inhibitors, this new PCR proved to be very advantageous. To demonstrate the reliability, buccal swabs from 2874 persons were typed not only with the new triplex PCR but also with a commercially available multiplex kit.     

Null allele can bring to interpretative problems in a deficitary paternity case

An STR null allele is an allele at a microsatellite locus that fails to amplify. A possible cause is poor primer annealing due to nucleotide sequence divergence in the flanking primers. In this study, a woman (ZAM) wanted to know whether a man (PGAF) was the father of her child (ZGC). During the court settlement, PGAF died. PGAF’s parents refused to undergo DNA investigation and denied the access to biological fragments from their dead son. Although, DNA specimens were obtained from buccal swabs of ZAM, ZGC and PGAF’s paternal sister (PTFS). Initially, only autosomal profiles were studied, and kinship assignment was inconclusive. Following our requests, PGAF’s parents (PRGF and LLGM) led us to obtain their DNA specimens. Only with the PTFS genetic profile, we were not able to demonstrate a kinship assignment. PTFS showed a homozygosis at D8S1179 locus. Then, merely comparing PTFS, LLGM and ZGC autosomal genetic profiles it was possible to underline that they were three different homozygous at D8S1179 locus. Hence, comparing the peak heights in different loci and according to literature, they had to carry a null allele at this locus. Parental studies were completed by Y haplotype analysis.     

Testing for genetic structure in different urban Argentinian populations

Fifteen autosomal short tandem repeat (STR) markers (D3S1358, HUMTH01, D21S11, D18S51, PENTA E, D5S818, D13S317, D7S820, D16S539, CSF1PO, PENTA D, HUMvWA, D8S1179, HUMTPOX, FGA) were analyzed in 1734 individuals living in urban areas of cities from six different Argentinian provinces (Buenos Aires, Neuquén, Tucumán, La Pampa, San Luis, Santa Cruz) in order to determine if a common urban database could be used in Argentina for forensic purposes. Frequencies estimates, Hardy-Weinberg equilibrium (HWE), and other parameters of forensic interest were computed. Comparisons between the six populations, and with published data from one Native American population from Argentina and other urban populations from Argentina and Europe were also performed. Our results reveal evidences for population structure, both when testing for genetic differentiation and when comparing frequencies distributions between different pairs of populations. Therefore, caution should be taken when using a common pooled database with general forensic purposes in Argentina.     

STR复合扩增及荧光检测技术在个体识别中的应用

目的 :使用 310型遗传分析仪对D3S1358等 10个位点进行基因型检测并应用于法医物证学个体识别案件。方法 :用PCR复合扩增结合四色荧光检测技术对样本DNA进行基因分型。结果 :常见物证检材可成功地得到检验。结论 :这些位点适用于法医物证学个体识别。     

Thai population data on 15 tetrameric STR loci-D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA

The genetic variations for 15 short tandem repeat (STR) loci-D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA, were performed on a population of 210 unrelated Thai individuals using the commercially available AmpF/STR Identifiler kit.     

Allele frequencies of 19 STR loci in a Philippine population generated using AmpFlSTR multiplex and ALF singleplex systems

Allele frequencies for the 19 short tandem repeat (STR) loci CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S306, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, DHFRP2 (FOLP23), F13A01, FES/FPS, FGA, TH01, TPOX, and vWA were obtained from a sample of 106 unrelated Filipinos from different regions of the Philippine archipelago.     

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.     

Allele frequencies of 15 STR loci using AmpF/STR Identifiler kit in a Korean population

The genetic variations for 15 short tandem repeat (STR) loci D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA were performed on 231 unrelated Korean population using commercially available AmpF/STR Identifiler kit.     

Validation and implementation of the PowerPlex 16 BIO System STR multiplex for forensic casework

The PowerPlex 16 BIO multiplex short tandem repeat (STR) system contains the 13 CODIS loci (FGA, TPOX, D8S1179, vWA, D18S51, D21S11, TH01, D3S1358, CSF1PO, D16S539, D7S820, D13S317, and DS5S818), plus two pentanucleotide repeat loci (Penta D and Penta E) and the sex-identifying locus. Amelogenin. The PowerPlex 16 BIO System is optimized for use with the Hitachi FMBIO gel imaging systems. A consortium of seven independent laboratories collaborated to perform the studies defined by the FBI standards for performing a developmental validation, including the evaluation of sample concordance, percent stutter determination, nonprobative casework, precision, sensitivity, mixture determination, effect of substrates, the impact of environmental insults, and species specificity. All samples tested for concordance were consistent except for one sample from the Virginia Division of Forensic Science database that displayed discordance at D13S317, a locus whose primer sequence was altered. Stutter values were comparable to those of other STR multiplex systems, the precision was comparable to other multiplexes analyzed by gel electrophoresis, the DNA profiles were unchanged by the substrate upon which the blood samples were placed, and the nonprobative casework samples re-typed for the PowerPlex 16 BIO System were consistent with previous typing results. When greater than 0.125 ng of DNA was placed into the PowerPlex 16 BIO System amplification reaction, a full profile was generated by all laboratories. The mixture study results were comparable to those reported for other multiplex systems, the environmental study demonstrated a loss of larger molecular weight loci when samples were incubated at elevated temperatures for a prolonged period of time, and the only notable cross species hybridization was observed with primate DNA samples. This extensive validation work performed demonstrates that the PowerPlex 16 BIO System provides STR data of a quality comparable with other PowerPlex STR multiplex kits as well as other widely used STR multiplexes and is thus suitable for evidentiary casework analysis as well as database sample profiling.     

复合扩增18个基因座的初步研究

目的建立五色荧光18个基因座的复合扩增检验体系。方法设计、合成引物,通过调整引物浓度和复合扩增条件,建立起五色荧光复合扩增体系,包含Amelogen、D6S1043、D21S11、D7S820、CSF1PO、D2S1338、D3S1358、D13S317、D8S1179、D16S539、PentaE、TH01、TPOX、PentaD共计18个基因座。结果该复合扩增系统检验结果稳定,分型准确。结论五色荧光18个基因座复合扩增系统构建成功。     

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.     

Turkish population data with the CODIS multiplex short tandem repeat loci.

Allele frequencies for 13 tetrameric short tandem repeat (STR) loci, CSF1PO, D18S51, D3S1358, D21S11, D5S818, FGA, D7S820, HUMTH01, D8S1179, TPOX, D13S317, VWA, and D16S539 were determined on 198 Turkish blood samples.     

Population data on the AmpF/STR Identifiler loci in Africans and Europeans from South Africa

Allele frequencies of 15 short tandem repeat (STR) loci, D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA, were determined for 98 unrelated Africans from South Africa and 98 unrelated Europeans from South Africa using the AmpFlSTR Identifiler PCR amplification kit. The genotype frequency distributions of the 15 STR loci were in the Hardy-Weinberg equilibrium for both populations.     

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.     

D5S818 Typing Discrepancy Between PowerPlex® Fusion and Other STR Kits Including GlobalFiler® Caused by a One‐base Deletion in 31 Nucleotides Upstream of the Repeat Region

Short tandem repeat (STR) typing is widely used in forensic investigation. When the same DNA sample is analyzed with different STR typing kits, a typing discrepancy is occasionally observed. In this study, we examined the cause of a typing discrepancy in a sample at D5S818 locus. This sample was designated as 10, 12 using Identifiler^®, Identifiler^® Plus, GlobalFiler^®, PowerPlex^® 16HS, and PowerPlex^® 18D, but as 9.3, 12 using PowerPlex^® Fusion. Sequencing results indicated that the shorter allele in the sample had a deletion (U31Tdel) at 31 nucleotides upstream of the repeat region (AGAT)₁₀. This deletion was located in the binding site of the published D5S818 forward primer in PowerPlex^® 16 and was only 9 and 11 nucleotides downstream of our estimated 5′ end position of D5S818 forward primer in GlobalFiler^® and PowerPlex^® 18D, respectively. We also examined the effect of primer length on the heterozygous peak balance in this sample.     

STR位点D19S253和D8S1179的法医学意义及应用研究

为评估STR位点D19S253和D8S1179的法医学应用价值，应用PCR和PAG垂直电泳技术对两位点的种属特异性，检测灵敏度，以及同一个体不同组织分型的同一性及不同基质和不同保存时间的斑痕分型等与法医应用有关的问题进行了研究，D19S253和D8S1179位点的检测灵敏度分别为0．25ng及0．5ng，同时两位点具有较高的种属特异性，同一性及较好重复性，且能够复合扩增，表明D19S253和D8S1179是法医学检案中较实用的两个STR标记。