Performance evaluation of two multiplexes used in fluorescent short tandem repeat DNA analysis

The performance of two commercial multiplex kits that together amplify the 13 core short tandem repeat (STR) loci currently in use by forensic laboratories and the U.S. national Combined DNA Indexing System (CODIS) were evaluated. The typing systems examined were AmpFlSTR Profiler Plus and AmpFlSTR COfiler (PE Applied Biosystems, Foster City, CA). Electrophoretic separation and detection of the fluorescent PCR products was achieved by capillary electrophoresis (CE) using an ABI Prism 310 Genetic Analyzer. The studies addressed the on-site validation of the instrument, the software, and each typing system. These studies included instrument sensitivity, resolution, precision, binning, peak height ratios, mixtures, stutter, and the amplification of non-probative and simulated forensic samples. Other additional developmental-type work is also reported herein, such as species specificity testing and amplification of environmentally insulted samples. Amplification conditions were found to be robust and the primer sets shown to be specific to human DNA. Stutter and peak height ratios fell within limits published by the manufacturer and other laboratories. The data demonstrate that the CE instrument can consistently resolve fragments differing in length by one base and that the +/-0.5 base bin used by the Genotyper software is acceptable for making accurate allele calls. Correct typing results were obtained from non-probative and simulated case samples, as well as samples exposed to outdoor environmental conditions. The results support the conclusion that DNA extracted from biological samples routinely encountered in the forensic laboratory can be reliably analyzed with AmpFlSTR Profiler Plus and COfiler using CE.     

AmpFlSTR profiler Plus short tandem repeat DNA analysis of casework samples, mixture samples, and nonhuman DNA samples amplified under reduced PCR volume conditions (25 microL)

As part of the validation of the AmpFlSTR Profiler Plus short tandem repeat (STR) system, under reduced polymerase chain reaction (PCR) volume conditions (i.e., 25 microL), a total of 275 casework samples were processed. Examples of profiles are presented along with amplification conditions to improve the odds of obtaining balanced and complete profiles for samples showing partial results or profiles with a descending slope. Data collected and used to develop our interpretation guidelines are included. From the mixture studies, full profiles were obtained for minor contributors, using 2 ng of DNA, with ratios of 10:1 or 1:20 and using 1 ng of DNA, with ratios of 10:1 and 1:8. The specificity of the Profiler Plus amplification reaction performed in 25 microL was examined and confirmed using a large spectrum of nonhuman DNAs. This report supports the use of the AmpFlSTR Profiler Plus STR system for casework DNA typing under reduced PCR volume conditions.     

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.     

Rapid and high-throughput forensic short tandem repeat typing using a 96-lane microfabricated capillary array electrophoresis microdevice

A 96-channel microfabricated capillary array electrophoresis (muCAE) device was evaluated for forensic short tandem repeat (STR) typing using PowerPlex 16 and AmpFlSTR Profiler Plus multiplex PCR systems. The high-throughput muCAE system produced high-speed <30-min parallel sample separations with single-base resolution. Forty-eight previously analyzed single-source samples were accurately typed, as confirmed on an ABI Prism 310 and/or the Hitachi FMBIO II. Minor alleles in 3:1 mixture samples containing female and male DNA were reliably typed as well. The instrument produced full profiles from sample DNA down to 0.17 ng, a threshold similar to that found for the ABI 310. Seventeen nonprobative samples from various evidentiary biological stains were also correctly typed. The successful application of the muCAE device to actual forensic STR typing samples is a significant step toward the development of a completely integrated STR analysis microdevice.     

Analysis of human fecal material for autosomal and Y chromosome STRs

Human stool samples from eight volunteers were stored under various conditions and extracted by three different procedures. Fecal material and tissue paper soiled with fecal material obtained from a crime scene were also extracted. Extracted DNA was amplified using the AmpFlSTR Profiler Plus, AmpFlSTR COfiler, and the AmpFlSTR Identifiler PCR amplification kits for the detection of the autosomal STR allelic patterns. DNA extracted from the male volunteers and from the soiled tissue paper evidence sample was also amplified using the Y-PLEX 6 and Y-PLEX 5 amplification kits. Analysis of the amplified products was carried out by capillary electrophoresis on the ABI PRISM 310 Genetic Analyzer. Autosomal and Y-STR profiles obtained from the fecal material were concordant with the results from the donors' buccal swabs.     

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.     

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.     

A time course study on STR profiles derived from human bone, muscle, and bone marrow.

The use of the polymerase chain reaction (PCR) to define deoxyribonucleic acid (DNA) types at several loci was investigated. PCR was used to amplify nine short tandem repeat (STR) loci along with the amelogenin locus on the X and Y chromosomes using the AmpF/STR Profiler Plus PCR amplification kit (Perkin Elmer). Rib bones were collected from 12 individuals. Five cm portions were buried at a depth of approximately 30 cm and 5 cm portions were left on the surface of the ground. Samples were exposed to the environment for periods of time ranging from two weeks to 17 months. Dried blood standards were prepared for use as reference standards for each rib sample. Bone, muscle, and bone marrow were collected from each sample. DNA from each tissue type was extracted. Complete profile results were obtained from the surface bone samples out to an exposure time of 17 months. None of the muscle or bone marrow samples produced complete profile results beyond eight weeks. All DNA typing results from complete or incomplete profiles were consistent with DNA typing results of the corresponding blood standard. Results suggest that using the AmpF/STR Profiler Plus PCR Amplification Kit is a valid way to establish the DNA profile of tissue types from human remains.     

Swiss Caucasian population data for the STR loci D2S1338 and D19S433 using the AmpFISTR SGM plus PCR amplification kit

Allele and genotype frequencies for the ten STR loci D3S1358, VWA, D16S539, D2S1338, D8S1179, D21S11, D18S51, D19S433, TH01, FGA were determined in a Swiss Caucasian population sample (n=206) using the AmpFISTR SGM Plus Amplification kit. Electrophoresis was carried out on an ABI PRISM CE 310 Genetic Analyzer instrument. Previously, allele frequencies were published for the 13 STR loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, THO1, TPOX, CSF1PO and D16S539 for the same samples (n=206) amplified with the AmpFISTR Profiler Plus and Cofiler PCR Amplification kits. Since the results for the eight loci D3S1358, VWA, FGA, D8S1179, D21S11, D18S51, THO1, D16S539 shared between the AmpFISTR SGM Plus, Profiler Plus and Cofiler PCR Amplification kits already are published, only the allele frequencies for the two STR loci D2S1338 and D19S433 are reported in this paper. The two loci meet Hardy-Weinberg expectations. In addition, there is little evidence for association of alleles among the 15 loci (amplified with the Profiler, Cofiler, and SGM Plus amplification kits). The allelic frequency data can be used in forensic analyses to estimate the frequency of a multiple STR locus DNA profile in the Swiss population.     

广西苗族人群15个STR基因座的多态性调查

目的调查广西苗族人群无关个体的15个STR基因座(D8S1179、D21S11、D7S820、CSF1PO、D3S1358、TH01、D13S317、D16S539、D2S1338、D19S433、vWA、TPOX、D18S51、D5S818、FGA)多态性,研究其在法医学检验中的应用价值。方法应用AmpFlSTRIdentifilerTM荧光标记复合扩增系统对274例广西苗族无关个体血样DNA进行15个STR基因座的复合扩增,用ABI3100遗传分析仪对扩增产物进行检测,用GeneScan、GenoTyper软件进行基因分型,统计计算15个STR基因座的群体遗传学参数。结果IdentifilerTM荧光标记系统的15个STR基因座在广西苗族人群的累积偶合率为5.04×10-17,累积非父排除率分别为0.9999993。结论该15个STR基因座可满足广西苗族人群法医学的个体识别及亲权鉴定的需要。     

The successful DNA typing of samples following a thermal cycler power loss

An approach for generating DNA profiles when critical samples have been consumed and a power outage occurs during the polymerase chain reaction (PCR) amplification reaction is described. This study demonstrates that a complete and accurate DNA short tandem repeat profile can be obtained: (1) when single source DNA samples are amplified for 26, 27, or 28 cycles using the Profiler Plus and COfiler Amplification Kits after an interruption in amplification, (2) from mock samples when PCR amplification has been interrupted early (after five cycles) or late (after 18 cycles) and the sample is subjected to an additional round of amplification, even after incubation of the sample at room temperature overnight, and (3) from nonprobative casework samples interrupted after approximately 18 cycles of amplification, an overnight incubation at room temperature and subjected to one or two additional rounds of PCR amplification for approximately 26 total cycles. Samples interrupted before five completed cycles and subjected to additional PCR cycles yielded variable results.     

尿液及尿斑DNA分型的研究

目的研究尿液及尿斑的DNA提取及其检验。方法用Chelex100法及QIAampMiniKit提取尿液及尿斑样本中的DNA,进行PCR扩增及STR检验。结果新鲜的及存放时间在12h以内的尿液样本能得到较好的分型结果;存放2d左右的尿液样本有50%能检出基因型;存放7d及更长时间的尿液样本全部不能检出基因型;尿斑样本的分型成功率很低。结论较新鲜的尿液样本均能进行DNA分型,在法医检案中具有应用价值。     

广西地区3个群体15个STR基因座频率多态性

目的调查广西地区仡佬族、仫佬族、瑶族无关个体的15个STR基因座(D8S1179、1321S11、D7S820、CSF1PO、D3S1358、TH01、D13S317、D16S539、D2S1338、D19S433、vWA、TPOX、D18S51、D5S818、FGA)多态性,研究其在法医学检验中的应用价值。方法 应用AmpFISTR IdentifilerTM荧光标记复合扩增系统,对314例仡佬族、332例仫佬族、238例瑶族无关个体血样DNA进行15个STR基因座的复合扩增,用ABI 3100遗传分析仪对扩增产物进行检测,GeneScan、GenoTyper软件进行基因分型,统计计算15个STR基因座的群体遗传学参数。结果IdertifilerTM系统的15个STR基因座在仡佬、仫佬、瑶族的累积偶合率为1.839×10-16~5.073×10-17,累积非父排除率为0.9999983~0.9999991。结论该15个STR基因座足可满足仡佬族、仫佬族、瑶族法医学的个体识别及亲权鉴定的需要。     

Recovery of genomic DNA from archived PCR product mixes for subsequent multiplex amplification and typing of additional loci: forensic significance for older unsolved criminal cases

A method for genomic DNA recovery from different types of PCR product mixes suitable for multiplex amplification and typing using the Profiler Plus STR typing system has been investigated. The application of this method is of significance in cases where the original DNA samples have been exhausted due to repeated typing analyses in an effort to maximize their evidentiary value. Such cases typically involve samples analyzed using the available DNA typing systems of the time which gave a markedly lower power of discrimination, either alone or in combination, compared to that of modern multiplex STR typing systems. It was found that an effective method for recovering genomic DNA from HLA-DQA1 +PM and CTT triplex amplification mixes, suitable for reproducible achievement of the complete Profiler Plus profile, involved the use of Amicon Microcon-100 microconcentrators. Interestingly, this method was not required to achieve the complete nine STR profile using D1S80 amplification mixes.     

烟蒂DNA分型的研究

目的 研究烟蒂中DNA提取及其检验。方法 用Chelex-100法提取170枚烟蒂样本的DNA,进行PCR扩增及STR检验。结果 除1名志愿者提供的21枚烟蒂外层纸未能检出STR基因分型外,其余烟蒂外层纸均得到分型结果。加入少许烟丝的样本未能检出STR分型,与口唇接触的海绵有时可检出基因型,6个月内的烟蒂可检出小片段基因座。结论 烟蒂能进行DNA分型,在法医检案中具有应用价值。     

Validation of STR typing by capillary electrophoresis

With the use of capillary electrophoresis (CE), high-resolution electrophoretic separation of short tandem repeat (STR) loci can be achieved in a semiautomated fashion. Laser-induced detection of fluorescently labeled PCR products and multicolor analysis enable the rapid generation of multilocus DNA profiles. In this study, conditions for typing PCR-amplified STR loci by capillary electrophoresis were investigated using the ABI Prism 310 Genetic Analyzer (Applied Biosystems). An internal size standard was used with each run to effectively normalize mobility differences among injections. Alleles were designated by comparison to allelic ladders that were run with each sample set. Multiple runs of allelic ladders and of amplified samples demonstrate that allele sizes were reproducible, with standard deviations typically less than 0.12 bases for fragments up to 317 bases in length (largest allele analyzed) separated in a 47 cm capillary. Therefore, 99.7% of all alleles that are the same length should fall within the measurement error window of +/- 0.36 bases. Microvariants of the tetranucleotide repeats were also accurately typed by the analytical software. Alleles differing in size by one base could be resolved in two-donor DNA mixtures in which the minor component comprised > or = 5% of the total DNA. Furthermore, the quantitative data format (i.e., peak amplitude) can in some instances assist in determining individual STR profiles in mixed samples. DNA samples from previously typed cases (typed for RFLP, AmpliType PM+DQA1, and/or D1S80) were amplified using AmpFlSTR Profiler Plus and COfiler and were evaluated using the ABI Prism 310. Most samples yielded typable results. Compared with previously determined results for other loci, there were no discrepancies as to the inclusion or exclusion of suspects or victims. CE thus provides efficient separation, resolution, sensitivity and precision, and the analytical software provides reliable genotyping of STR loci. The analytical conditions described are suitable for typing samples such as reference and evidentiary samples from forensic casework.     

Identification of a novel polymorphism in the X-chromosome region homologous to the DYS456 locus

During an extensive multipopulation study with Y-short tandem repeat (STR) loci, amplified using the AmpFlSTR Yfiler PCR amplification kit, amplification of a 71 bp fragment was observed in 2.32% of the male samples analyzed (N = 3141). By direct sequencing of this fragment, it was determined that the primer binding sequences were identical to those of the DYS456 locus. A T to G single-nucleotide polymorphism (SNP) enabled amplification of the 71 bp fragment. The SNP is located within an X-Y homologous region at Xq21.31 and was observed with the highest frequency within the African American and Sub-Saharan African populations in our study. Presence of SNP on the X chromosome did not interfere with the reliability of typing the DYS456 locus and the other Y-STR loci typeable using the AmpFlSTR Yfiler PCR amplification kit. Full profiles in a mixture of male:female at 1:4000 were obtained using the current configuration of the AmpFlSTR Yfiler kit even in the presence of female DNA containing the G variant.     

多重置换扩增技术用于法医学微量DNA检测效果

目的探讨多重置换扩增(MDA)技术对法医学微量DNA样品STR检测分型的效果。方法用MDA技术对不同模板量DNA进行全基因组扩增(WGA),扩增产物用实时荧光定量PCR技术定量、用Profiler PlusTM试剂盒检测基因型。结果该方法可对模板DNA增加104～106倍。1ng样品DNA的MDA产物可获得9个STR基因座和Amelogenin性别基因座的准确分型结果;低于0.1ng的样品DNA经MDA扩增后,基因座检出数增加,但可见等位基因不平衡或丢失现象。结论MDA技术可有效增加DNA模板量和提高微量DNA分型效果。但样品DNA量低于0.1ng时,MDA产物的STR分型结果判读须慎重。     

Systematic analysis of stutter percentages and allele peak height and peak area ratios at heterozygous STR loci for forensic casework and database samples

To assist the interpretation of STR DNA typing results from forensic casework samples containing mixtures, the range of heterozygous allele peak height and peak area ratios (HR) and stutter percentages (stutter %) for the loci comprised in the AmpFlSTR Profiler Plus (PP) kit were assessed on 468 database and 275 casework single source samples. Stutter % medians were similar for database and casework samples, ranging from 2% to 7%. The upper limit of the stutter value range was 16%, calculated as median +3 SD, although lower locus-specific values could be used. HR medians were 93 +/- 6.5% for database samples, 88 +/- 12% for casework samples. For casework samples, the maximum signal imbalance noted was 52%, calculated as median -3 SD. No significant difference was observed between peak height and peak area calculated values. This study shows the importance of selecting the proper reference database for the establishment of HR threshold values.     

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.