STR melting curve analysis as a genetic screening tool for crime scene samples

In this proof-of-concept study, high-resolution melt curve (HRMC) analysis was investigated as a postquantification screening tool to discriminate human CSF1PO and THO1 genotypes amplified with mini-STR primers in the presence of SYBR Green or LCGreen Plus dyes. A total of 12 CSF1PO and 11 HUMTHO1 genotypes were analyzed on the LightScanner HR96 and LS-32 systems and were correctly differentiated based upon their respective melt profiles. Short STR amplicon melt curves were affected by repeat number, and single-source and mixed DNA samples were additionally differentiated by the formation of heteroduplexes. Melting curves were shown to be unique and reproducible from DNA quantities ranging from 20 to 0.4 ng and distinguished identical from nonidentical genotypes from DNA derived from different biological fluids and compromised samples. Thus, a method is described which can assess both the quantity and the possible probative value of samples without full genotyping.     

DNA Typing for the Identification of Old Skeletal Remains from Korean War Victims*

Abstract: The identification of missing casualties of the Korean War (1950–1953) has been performed using mitochondrial DNA (mtDNA) profiles, but recent advances in DNA extraction techniques and approaches using smaller amplicons have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Therefore, 21 skeletal remains of Korean War victims and 24 samples from biological relatives of the supposed victims were selected based on circumstantial evidence and/or mtDNA‐matching results and were analyzed to confirm the alleged relationship. Cumulative likelihood ratios were obtained from autosomal short tandem repeat, Y‐chromosomal STR, and mtDNA‐genotyping results, and mainly confirmed the alleged relationship with values over 10⁵. The present analysis emphasizes the value of mini‐ and Y‐STR systems as well as an efficient DNA extraction method in DNA testing for the identification of old skeletal remains.     

Developmental Validation of the PrepFiler™ Forensic DNA Extraction Kit for Extraction of Genomic DNA from Biological Samples*

Abstract: The PrepFiler? Forensic DNA Extraction Kit enables isolation of genomic DNA from a variety of biological samples. The kit facilitates reversible binding of DNA with magnetic particles resulting in high DNA recovery from samples with very low and high quantities of biological materials: 0.1 and 40 μL of human blood (donor 2) provided 14 and 2883 ng of DNA, respectively. Following the revised SWGDAM guidelines, performance of the developed method was investigated using different sample types including saliva on swabs, semen stains on cotton fabric, samples exposed to environment, samples with polymerase chain reaction (PCR) inhibitors, blood stains (on denim, cotton cloth, and FTA^® paper), and touch evidence‐type samples. DNA yields for all samples tested were equal or better than those obtained by both phenol–chloroform extraction and commercial kits tested. DNA obtained from these samples was free of detectable PCR inhibitors. Short tandem repeat profiles were complete, conclusive, and devoid of PCR artifacts.     

Incestuous paternity detected by STR-typing of chorionic villi isolated from archival formalin-fixed paraffin-embedded abortion material using laser microdissection

Microscopic examination of a blood clot expelled by a physically and mentally disabled woman taken to the emergency room because of genital bleeding revealed the presence of chorionic villi encircled by decidua, hemorrhage, and necrosis. In order to identify the father of the product of conception, sections of formalin-fixed, paraffin-embedded abortion material were subjected to laser microdissection: DNA extraction from chorionic villi selectively isolated from the surrounding tissues allowed successful STR-typing of fetal cells, which was otherwise prevented by excess maternal DNA. The large number of homozygous genotypes in the fetal profile suggested incestuous paternity. Analysis of reference DNA samples from male relatives excluded the woman's father, paternal grandfather, and maternal grandfather, whereas the obligate paternal alleles of the fetus were constantly present in the genotypes of the woman's brother, clearly demonstrating brother-sister incest (probability of paternity > 99.99999%).     

Forensic DNA Analysis of Wheat Flour as Commonly Used in White Powder Cases

In the wake of terrorist attacks using anthrax and ricin, white powder is often encountered in cases of malicious mischief and terrorist threats. Wheat flour is a common white powder encountered in such criminal investigations. We used DNA analysis to investigate wheat flour samples for identification and discrimination as trace evidence. Species identification of commercially available wheat flour was carried out by sequencing a partial region of the ribulose bisphosphate carboxylase large subunit gene (rbcL). Samples were discriminated using short tandem repeat (STR) analysis. The rbcL sequences of all wheat flour samples were identical and showed a high level of similarity to known wheat (Triticum aestivum L.) sequences. Furthermore, flours had characteristic patterns in STR analyses, with specific cultivars showing distinctive patterns. These results suggested that the identification of wheat flour species is possible using rbcL sequencing, and that STR analysis is useful for discriminating between samples.     

An Optimized DNA Analysis Workflow for the Sampling,Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints

DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints—touch DNA “sandwiched” between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post‐amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri‐Sep? columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7–100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases.     

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.     

Haplotypic blocks of X-linked STRs for forensic cases: study of recombination and mutation rates

In complex kinship cases, markers situated in haplotypic blocks may provide additional clues to other unlinked markers. We have established a protocol to amplify six X-chromosome microsatellites, located in two haplotype blocks, using PCR with fluorochrome-labeled primers and capillary electrophoresis. The segregation stability was explored in 92 unrelated families with individuals from three generations. Sixty-one different haplotypes were found in the DXS10079-DXS10074-DXS10075 block in the grandfathers and 96 in the mothers, with estimated haplotype diversities of 0.9828 and 0.9842, respectively. Fifty and 73 different haplotypes were found in the DXS6801-DXS6809-DXS6789 block in the grandfathers and the mothers, with estimated haplotype diversities of 0.9711 and 0.9742, respectively. We observed 10 between-cluster and one within-cluster recombinations in 99 female meioses. The overall per-locus mutation rate was 0.0034. This protocol allows for the characterization of the alleles of two sets of linked markers of the X-chromosome that can be useful in complex forensic cases.     

A D19S433 primer binding site mutation and the frequency in Japanese of the silent allele it causes

Short tandem repeat studies are powerful tools for parentage analysis and for identification of missing persons, victims of murder, and victims of mass fatalities when reference samples are unavailable. The primer in the Identifiler kit failed to amplify an allele at the D19S433 locus, producing a silent ("null") allele. The causal mutation is a base change (G>A) 32 nucleotides downstream from the 3' end of the AAGG repeats. The silent alleles are problematical in parentage analysis because when transmitted, they can cause a parent-child inconsistency that is unrelated to Mendelian genetics. The inconsistency is sometimes termed an "apparent opposite homozygosity" and it produces false evidence of nonparentage. Alternative primers were designed to amplify the D19S433 locus alleles and they detect the silent allele. Frequencies of the (no longer) silent allele were determined to be 0.0114 in 176 people from Shizuoka (Honshu) and 0.0128 in 156 people from Okinawa.     

A Comparison of DNA Extraction Using AutoMate Express™ and EZ1 Advanced XL from Liquid Blood,Bloodstains, and Semen Stains

In this study, DNA was extracted using an AutoMate Express? and an EZ1 Advanced XL from liquid blood, fresh and aged bloodstains, and fresh and aged semen stains. Extracted DNA was quantified by real‐time PCR using the D17Z1 locus. Short tandem repeat typing was performed using an AmpF?STR^® Identifiler kit. The yields of DNA obtained by the AutoMate Express? were higher from fresh bloodstains and fresh semen stains, almost the same from aged bloodstains and aged semen stains, but slightly lower from liquid blood compared with those obtained by the EZ1 Advanced XL. The addition of dithiothreitol or the use of PrepFiler? lysis buffer improved the EZ1 Advanced XL results from fresh bloodstains, but not for liquid blood and aged bloodstains. Our results demonstrated that the PrepFiler? lysis buffer is the main contributor to the higher DNA yields of the AutoM ate Express? for fresh bloodstains.     

中国28个省/区汉族人群41个STR基因座多态性数据分析

目的调查分析中国28个省/区汉族人群41个STR基因座的遗传多态性,为相关研究和鉴定提供全面科学的基础数据。方法收集28个省/区汉族9 126名无关个体血样本,采用Global FilerTM、AGCU EX-22和AGCU 21+13种试剂盒,进行41个STR基因座分型,统计各基因座等位基因分布和遗传学参数,并对各数据之间进行差异性检验。结果中国28个省/区汉族人群41个基因座分别检出8~76个等位基因,各基因型分布经正和检验,除SE33等7个基因座外,均符合Hardy-Weinberg平衡定律。遗传学参数分析显示,41个基因座中SE33等21个属高鉴别力、高杂合度、高信息量,其余20个属中等高度多态性基因座。部分省份等位基因频率分布数据之间有显著性差异(P0.05),各省与全国综合数据之间则无差异(P0.05)。结论本文调查结果证实41个STR基因座均具有较强的个体识别能力,获得的数据可为法医学应用和筛选适合中国汉族人群基因座等研究和实践提供科学的基础数据。     

Determination of Both Fetus’ and Mother's Blood Type from an Autopsy Case Immersed in Formalin for Over 50 Years

A female fetus which had been immersed in formalin for more than 50 years was found in Japan. Because no liquid blood could be obtained, we tried to use immunohistochemistry (IHC) methods to tissue samples obtained at autopsy to identify both the fetal and mother's blood type. We detected B antigens on endothelial cells in paraffin sections of the fetal organs. Furthermore, we observed both anti‐A‐ and anti‐B‐positive red blood cells in the intervillous space, which is indicative of the mother's blood type. To our knowledge, this is the first case report on determining the blood type of both the fetus and the mother from tissue immersed in formalin for such a long time. The results suggest that IHC is valuable for the determination of ABO blood type in circumstances of long postmortem duration and unfavorable storage conditions.     

海南汉族人群19个STR基因座遗传多态性及其应用

目的建立海南地区汉族人群19个常染色体STR基因座的遗传多态性数据资料,并探讨此19-STR基因座系统在亲子鉴定中的应用。方法对海南汉族462例无血缘关系个体,采用Goldeneye~(TM) 20A系统复合扩增并检测,得到19个STR基因座的遗传数据信息;在283例亲子鉴定案例中,评价19-STR基因座系统的应用。结果 19个STR基因座的基因频率分布均符合Hardy-Weinberg平衡(P0.05),杂合度在0.603~0.914之间,累积个体识别率大于0.999 999 999 999 999,累积三联体非父排除率为0.999 999 994。283例亲子鉴定中,三联体170例,二联体113例;认定案例247例(87.3%),排除案例36例(12.7%);发生等位基因突变案例14例(4.9%),均为一步突变。结论 19个STR基因座中的14个基因座具有高度遗传多态性,19-STR基因座复合扩增分型系统具有较高的非父排除效能,可满足海南地区亲子鉴定的需要,同时应注意亲子鉴定中的基因突变现象。     

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.     

A Cannabis sativa STR Genotype Database for Australian Seizures: Forensic Applications and Limitations*

Abstract: A genetic database was established with the aim of documenting the genetic diversity of Cannabis sativa in Australia for future utilization in forensic investigations. The database consisted of genotypes at 10 validated short tandem repeat loci for 510 plants representing drug seizures from across Australia and 57 fiber samples. A total of 106 alleles and 314 different genotypes were detected. All fiber samples exhibited unique genotypes while 55% of the drug samples shared a genotype with one or more samples. Shared genotypes were mostly found within seizures; however, some genotypes were found among seizures. Statistical analysis indicated that genotype sharing was a consequence of clonal propagation rather than a lack of genetic resolution. Thus, the finding of shared genotypes among seizures is likely due to either a common supplier, or direct links among seizures. Notwithstanding the potential intelligence information provided by genetic analysis of C. sativa, our database analysis also reveals some present limitations.     

Analysis of Y-STR loci in a population sample from northeast China

POPULATION: A total of 141 unrelated Chinese Han male individuals living in Liaoning in northeast China.