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。     

The First Successful Use of a Low Stringency Familial Match in a French Criminal Investigation

We describe how a very simple application of familial searching resolved a decade‐old, high‐profile rape/murder in France. This was the first use of familial searching in a criminal case using the French STR DNA database, which contains approximately 1,800,000 profiles. When an unknown forensic profile (18 loci) was searched against the French arrestee/offender database using CODIS configured for a low stringency search, a single low stringency match was identified. This profile was attributed to the father of the man suspected to be the source of the semen recovered from the murder victim Elodie Kulik. The identification was confirmed using Y‐chromosome DNA from the putative father, an STR profile from the mother, and finally a tissue sample from the exhumed body of the man who left the semen. Because of this identification, the investigators are now pursuing possible co‐conspirators.     

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.     

Validation and casework testing of the BioPlex-11 for STR typing of telogen hair roots

A new STR typing strategy has been developed allowing the simultaneous amplification and subsequent analysis of 11 polymorphic systems with amplicon sizes smaller than 270bp. The multiplex amplification reaction includes six STR loci from the European standard set of loci (ESS) for DNA databases (D3S1358, D8S1179, D21S11, THO1, FGA and VWA) as well as four additional STR systems selected for their robustness (D2S1338, D12S391, TPOX and D5S818) together with the sex-specific locus amelogenin. After PCR amplification, the multiplex reaction is splitted into two sets of STR multiplexes by using biotin labelled primers only for one set. Using streptavidin-coated Sepharose beads five STR systems are separated from the other six systems prior to being analysed in two different runs on a capillary gel electrophoresis instrument. The multiplex system was developed and tested especially for the use in forensic casework if only limited amounts or highly degraded DNA is available, for instance, when isolated from telogen hair roots.     

亲权鉴定中常用STR基因座的基因组学和遗传学分析

美国联邦调查局建立的联合DNA索引系统即CODIS系统至今已有10年,该系统确定的13个STR基因座及近年来开发的PentaD、PentaE、D2S1338和D19S433基因座被全世界范围内的实验室广泛采用,在亲权鉴定和罪犯数据库建设等方面发挥了重要的作用。本文利用Web of Knowledge核心检索系统和Elsevier等全文数据库及网络资源,对近年来常用STR基因座的基因组学特征和遗传学特征进行了深入地分析,并对这些STR基因座在实践中的应用情况进行了综述。     

Validation of short tandem repeats (STRs) for forensic usage: performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples

The amplification and typing conditions for the 13 core CODIS loci and their forensic applicability were evaluated. These loci are CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11. Results were obtained using the multiplex STR systems AmpFlSTR Profiler Plus and AmpFlSTR COfiler (Applied Biosystems, Foster City, CA), GenePrint PowerPlex (Promega Corporation, Madison, WI), and subsets of these kits. For detection of fluorescently labeled amplified products, the ABI Prism 310 Genetic Analyzer, the ABI Prism 377 DNA Sequencer, the FMBIO II Fluorescent Imaging Device, and the Fluorlmager were utilized. The following studies were conducted: (a) evaluation of PCR parameter ranges required for adequate performance in multiplex amplification of STR loci, (b) determination of the sensitivity of detection of the systems, (c) characterization of non-allelic PCR products, (d) evaluation of heterozygous peak intensities, (e) determination of the relative level of stutter per locus, (f) determination of stochastic PCR thresholds, (g) analysis of previously typed case samples, environmentally insulted samples, and body fluid samples deposited on various substrates, and (h) detection of components of mixed DNA samples. The data demonstrate that the commercially available multiplex kits can be used to amplify and type STR loci successfully from DNA derived from human biological specimens. There was no evidence of false positive or false negative results and no substantial evidence of preferential amplification within a locus. Although at times general balance among loci labeled with the same fluorophore was not observed, the results obtained were still valid and robust. Suggested criteria are provided for determining whether a sample is derived from a single source or from more than one contributor. These criteria entail the following: (a) the number of peaks at a locus, (b) the relative height of stutter products, and (c) peak height ratios. Stochastic threshold levels and the efficiency of non-templated nucleotide addition should be considered when evaluating the presence of mixtures or low quantity DNA samples. Guidelines, not standards, for interpretation should be developed to interpret STR profiles in cases, because there will be instances in which the standards may not apply. These instances include (a) a primer binding site variant for one allele at a given locus, (b) unusually high stutter product, (c) gene duplication, and (d) translocation.     

Forensic and population genetic analysis of Serbian population using 21 STR loci of GlobalFiler™ PCR amplification kit

Autosomal short tandem repeats (STRs) have been widely used in forensic investigations. Prior to the application of any DNA based identification method, it is essential to estimate the allele frequencies and forensic statistical parameters of targeted STR loci in each population in order to provide a more precise reference database for forensic investigation. The GlobalFiler™ Kit is a multiplex assay that combines the 13 original CODIS loci with 7 non-overlapping loci from the expanded European Standard Set (ESS), as well as the highly discriminating SE33 locus, two Y-based loci and the sex determining maker, Amelogenin. The full complement of loci in the GlobalFiler™ Kit are: D13S317, D7S820, D5S818, CSF1PO, D1S1656, D12S391, D2S441, D10S1248, D18S51, FGA, D21S11, D8S1179, vWA, D16S539, TH01, D3S1358, AMEL, D2S1338, D19S433, DYS391, TPOX, D22S1045, SE33 and a Y-specific insertion/deletion locus (Yindel). The 6-dye GlobalFiler™ PCR Amplification kit (ThermoFisher Scientific) comprises 21 autosomal STRs have already been proven to be able to provide reliable DNA profiling results and enhance the power of discrimination between individuals. In this study, we are presenting an analysis of GlobalFiler STR loci on 209 unrelated individuals from Serbia.     

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.     

The development of reduced size STR amplicons as tools for analysis of degraded DNA

New multiplex PCR sets of commonly used short tandem repeat (STR) markers have been developed to produce PCR products that are reduced in size when compared to standard commercial STR kits. The reduction in size of these amplicons can facilitate the examination and analysis of degraded DNA evidence by improving amplification efficiency. This "miniSTR" approach will permit current forensic practitioners to use STR markers and instrumentation already present in their laboratories and to generate genotyping data that is directly comparable to reference samples and searchable through the FBI's Combined DNA Index System (CODIS) databases. This paper discusses the development of these new primer sets and presents some initial results in the analysis of degraded and aged DNA samples. A method for removal of problematic fluorescent dye artifacts is also described. Comparison studies in over 100 samples have verified that these miniSTR primers can provide fully concordant results to commercial STR kits and can provide improved signal from degraded DNA specimens. These miniplex sets should prove valuable in the analysis of samples where allele dropout and reduced sensitivity of larger STR alleles occurs.     

15个STR在单亲亲子鉴定中的排除分析

目的评估15个短串联重复(DODIS)基因座和Penta D、Penta E)在单亲亲子鉴定中的亲权排除率。方法 以644个随机个体构成207046个假想的家系,然后统计不排除或只有一个基因座排除亲权关系的例数。结果有27个假想家系15个STR均不排除亲权关系,排除率为0.999869。有384个假想家系只有一个基因座排除,占0.185％。结论用15个STR认定单亲案的亲权关系时要谨慎。出现单个基因座排除时要检测更多的遗传标记,以区分是否系突变。     

Maine Caucasian population DNA database using twelve short tandem repeat loci

A population study of Caucasians residing in Maine was conducted using the AmpF1STR Profiler PCR Amplification Kit and the AmpF1STR Profiler Plus PCR Amplification Kit (Applied Biosystems Division (ABD) of Perkin Elmer, Foster City, CA). The kits contain the reagents necessary to amplify 12 different STR loci and the gender marker Amelogenin using two multiplex PCR, each containing nine STR loci. Thus, there is an overlap of six STR loci. The 12 STR loci are TH01, TPOX, CSF1PO, D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, and D7S820. These loci represent 12 of the 13 core loci selected by the CODIS STR standardization project. Dye-labeled amplification products were separated and detected using the capillary electrophoresis instrument ABI Prism 310 Genetic Analyzer. Allele frequencies were determined for the 12 STR loci. Statistical analysis of the data included Hardy-Weinburg equilibrium (HWE) analysis, pairwise independence testing, power of discrimination (PD), and probability of exclusion (PE).     

Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples

An additional 20 novel mini-short tandem repeat (miniSTR) loci have been developed and characterized beyond the six previously developed by our laboratory for a total of 26 non-CODIS miniSTR markers. These new markers produce short PCR products in the target range of 50-150 base pairs (bp) by moving the primer sequences as close as possible-often directly next to the identified repeat region. These candidate loci were initially screened based on their small amplicon sizes and locations on chromosomes currently unoccupied by the 13 CODIS STR loci or at least 50 Mb away from them on the same chromosome. They were sequenced and evaluated across more than 600 samples, and their population statistics were determined. The heterozygosities of the new loci were compared with those of the 13 CODIS loci and all were found to be comparable. Only five of the new loci had lower values than the CODIS loci; however, all of these were much smaller in size. This data suggests that these 26 miniSTR loci will serve as useful complements to the CODIS loci to aid in the forensic analysis of degraded DNA, as well as missing persons work and parentage testing with limited next-of-kin reference samples.     

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.     

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.     

Results of the 2018 Rapid DNA Maturity Assessment

Three commercially available integrated rapid DNA instruments were tested as a part of a rapid DNA maturity assessment in July of 2018. The assessment was conducted with sets of blinded single-source reference samples provided to participants for testing on the individual rapid platforms within their laboratories. The data were returned to the National Institute of Standards and Technology (NIST) for review and analysis. Both FBI-defined automated review (Rapid DNA Analysis) and manual review (Modified Rapid DNA Analysis) of the datasets were conducted to assess the success of genotyping the 20 Combined DNA Index System (CODIS) core STR loci and full profiles generated by the instruments. Genotype results from the multiple platforms, participating laboratories, and STR typing chemistries were combined into a single analysis. The Rapid DNA Analysis resulted in a success rate of 80% for full profiles (85% for the 20 CODIS core loci) with automated analysis. Modified Rapid DNA Analysis resulted in a success rate of 90% for both the CODIS 20 core loci and full profiles (all attempted loci per chemistry). An analysis of the peak height ratios demonstrated that 95% of all heterozygous alleles were above 59% heterozygote balance. For base-pair sizing precision, the precision was below the standard 0.5 bp deviation for both the ANDE 6C System and the RapidHIT 200.     

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.     

Effectiveness of Coupled Application of AmpFℓSTR Yfiler Kit and Reduced Size Y‐chromosomal Short Tandem Repeat Analysis for Archeological Human Bones

The AmpF?STR Yfiler PCR Amplification (Yfiler) kit continues to be improved for a better analytical efficiency in cases of highly degraded DNA. The authors endeavored to determine whether coupling of the Yfiler kit with supplemental multiplex amplification of some Y‐STR loci is a more efficient analytical mode for poorly preserved human femurs (n = 15) discovered at Korean archeological sites. To reveal locus profiles not easily obtained by Yfiler analysis, custom‐designed primers were adopted for the DYS390, DYS391, DYS392, DYS438, DYS439, and DYS635 loci. The success rate for 16 Y‐STR locus profiles obtained from the 15 femurs was improved from 18.33% (in the use of Yfiler kit only) to 49.17% (the coupled use of Yfiler and custom‐designed primers). In this study, the authors established that the custom‐designed primers offer a markedly improved success rate for obtainment of Y‐STR profiles from degraded aDNA not easily identified by sole use of the Yfiler assay.     

Constructing universal multiplex PCR systems for comparative genotyping

Analysis of length polymorphisms at STR loci in the human genome has become a standard approach for comparative genotyping in many areas including disease research and diagnostics, parentage assessment, investigations of human diversity, and forensic science. The simultaneous analysis of multiple STR loci through multiplex PCR and multicolor fluorescence detection offers sample conservation, high throughput, and automated genetic analysis. Careful design and optimization of tetranucleotide STR multiplexes has led to reliable, standardized systems that powerfully differentiate and distinguish individual human DNA profiles. The development of these multiplex systems involved a rigorous experimental strategy that included careful selection of PCR primer sequences (for yield, specificity, and multiplex compatability), along with optimization of PCR component concentrations, thermal cycling parameters, and fluorescence detection conditions. This developmental approach rendered well-characterized DNA typing systems that are high performing (sensitive, specific, and balanced), optimized to universal parameters (same reaction conditions), resilient to fluctuations in reaction conditions, and simple to implement and use routinely.     

Informativeness of the CODIS STR loci for admixture analysis

Population admixture (or ancestry) is used as an approach to gene discovery in complex diseases, particularly when the disease prevalence varies widely across geographic populations. Admixture analysis could be useful for forensics because an indication of a perpetrator's ancestry would narrow the pool of suspects for a particular crime. The purpose of this study was to use Fisher's information to identify informative sets of markers for admixture analysis. Using published founding population allele frequencies we test three marker sets for efficacy for estimating admixture: the FBI CODIS Core STR loci, the HGDP-CEPH Human Genome Diversity Cell Line Panel and the set of 39 ancestry informative SNPS from the Shriver lab at Pennsylvania State University. We conclude that the FBI CODIS Core STR set is valid for admixture analysis, but not the most precise. We recommend using a combination of the most informative markers from the HGDP-CEPH and Shriver loci sets.     

Use of sibling pairs to determine the familial searching efficiency of forensic databases

Pairs of individuals tested at the 13 CODIS core STR loci to determine sibship were used as a source of familial data that was seeded into a larger data set of 12,000 plus DNA profiles simulating a CODIS-like offender database. To determine whether known sibs could be found in the larger database two methods were used: degree of allele sharing and a kinship matching approach. The allele sharing method detected 62 of 109 of the known sib pairs (57%) while kinship matching detected 90 of the sib pairs (83%). Although kinship matching was the more efficient method of the two, the number of false positives generated prior to finding a true match was inversely related to the likelihood of sibship suggesting that many true siblings would not be easily found in a large forensic database via familial searching techniques.