Validation of the InnoXtract™ DNA extraction method for bone, teeth, and rootless hair.

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.

Development and validation of a novel method "SpermX™" for high throughput differential extraction processing of sexual assault kits (SAKs) for DNA analysis.

The Sperm X method uses a nanotechnology derived polymer membrane that functions as a separation medium to effectively trap sperm cells while enabling efficient flow through of the digested epithelial cell DNA. This specialized membrane enabled development of a method that could significantly increase a forensic laboratory's ability to obtain male sperm fraction DNA profiles. The SpermX device provides a rapid, reproducible procedure that is easy to implement in a single-tube format as well as high-throughput truly automated hands-free workflows. Validation studies, performed using the manual SpermX method, include sensitivity, stability, precision (reproducibility and repeatability), mixtures, and a method comparison to the traditional differential extraction. Sensitivity and method comparison studies demonstrated a wide range of sperm cells, from a high of over 2.78 million cells (9158 ng) to a low of 25 cells (83 pg), can be trapped by the SpermX membrane. Stability studies on various substrates (i.e., carpet, cotton, denim, polyester, and silk) and degraded semen gave the expected male DNA profiles. Data from the same operator and a different operator were consistent with low variance. Mixtures, with ratios ranging from approximately 10:1-18182:1, created to simulate real casework type samples including buccal/semen, vaginal epithelial/semen, and post coital swabs at different time intervals, were tested. A comparison of the SpermX method to the conventional differential extraction method resulted in comparable probative male profile allelic data and associated statistical probabilities. For low level sperm samples, down to 25 sperm cells (83 pg), the SpermX method outperformed the conventional differential extraction with more genotypic information and associated probabilities.

Successful nuclear DNA profiling of rootless hair shafts: a novel approach.

Historically, rootless hair shaft samples submitted to a forensic laboratory for DNA analysis are reserved for mitochondrial DNA (mtDNA) analysis due to the presence of highly degraded as well as insufficient amounts of nuclear DNA. Although mtDNA has been very successful in obtaining results from rootless hair, this system has its limitations, namely, it is a lineage marker that cannot differentiate between maternally related genotypes. Given the high incidence of hairs as forensic evidence, there is a need for the use of a nuclear DNA test system capable of producing reliable results for hair shaft forensic evidence. This study reports the utilization of an enhanced DNA extraction methodology for hairs, in combination with a recently developed novel, nuclear DNA typing assay, InnoTyper® 21, to improve the success rate for obtaining informative results from highly compromised, degraded, and trace forensic samples such as rootless hair shafts. The InnoTyper 21 kit is a small amplicon retrotransposon marker typing system compatible with currently used capillary electrophoresis platforms. This system contains 20 Alu element markers, ranging in size from 60 to 125 bp, making the assay highly sensitive for extremely degraded forensic samples and thus enabling recovery of nuclear DNA profiles from samples that would otherwise require mtDNA sequencing. A subset of samples was also tested with the GlobalFiler kit with less success due to the larger amplicon sizes in comparison with InnoTyper 21. Results were variable but very promising, with approximately 40% of the total number of hairs tested producing interpretable nuclear DNA profiles with InnoTyper 21. These results demonstrate the ability of the utilized methodologies to produce nuclear DNA results with high statistical power from rootless hair shafts.

Development and validation of a novel multiplexed DNA analysis system, InnoTyper® 21.

We report here a novel multiplexed DNA analysis system consisting of 20 Alu markers and Amelogenin for analysis of highly degraded forensic biological samples. The key to the success of the system in obtaining results from degraded samples is the primer design yielding small amplicon size (60-125bp) for all 20 markers. The markers included in the InnoTyper® 21 system are bi-allelic, having two possible allelic states (insertion or null) and thus termed INNULs. The markers are short interspersed nuclear elements (SINEs), a category of retrotransposable elements (REs) which are non-coding genomic DNA repeat sequences, or "mobile insertion elements," comprising approximately 40% of the human genome. Alu elements are primate specific SINEs that have reached a copy number in excess of one million in the human genome, which makes these markers highly sensitive and desirable for forensic samples with extremely degraded DNA. Until now however, due to the inherent size difference associated with insertion and no insertion alleles, the use of Alu REs has not been practical for forensic applications. The novel primer design described herein has allowed the development of a multiplexed Alu system yielding fragment sizes amenable to degraded DNA samples, as frequently encountered in missing persons cases or forensic samples such as hair shafts. Although use of Alus in human identity has been studied using single marker amplification and reported before, we report for the first time development and validation of a system with multiplexed RE markers. Studies performed include PCR optimization, species specificity, sensitivity, degradation and inhibition, precision and accuracy, nonprobative samples, mixture, and population database studies. A population study using 592 samples including five populations was performed using InnoTyper 21. The data indicated the random match probability for the combination of these 20 Alu markers was greater than 1 in 3.8 million for the populations studied, indicating the greater statistical power of these autosomal nuclear DNA markers over haplotype systems typically used in such degraded samples. Results demonstrate the system is successful in obtaining results from highly degraded DNA. A sensitivity study performed demonstrated at least 95% recovery of alleles from as low as 50pg of total input DNA, and partial profiles from as low as 25pg. This study has demonstrated that the bi-allelic INNULs in the InnoTyper 21 system provide a sensitivity of detection and a power of discrimination that makes them useful for human identification of extremely degraded samples.

Stable Tricyclic Antitubercular Ozonides Derived from Artemisinin.

New, highly stable tricyclic antitubercular ozonides 9 and 10 derived from artemisinin are reported in 39 and 9% yields, respectively. The ozonide groups of 9 and 10 were found to be stable under strong basic and acidic conditions. The absolute configuration of ozonides 9 was confirmed by X-ray crystallography. Ozonide 10 shows promising antitubercular activity against M. tuberculosis H37Ra and M. tuberculosis H37Rv with MIC values of 0.39 and 3.12 µg/mL, respectively.

Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.

There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample. Alu are Short Interspersed Elements (SINE), approximately 300bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80bp "short" DNA fragment and a 207bp "long" DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a "Degradation Index", or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss of STR markers and alleles as degradation increases. Use of a synthetic target as an internal positive control (IPC) provides an additional assessment for the presence of PCR inhibitors in the test sample. In conclusion, a DNA based qualitative/quantitative/inhibition assessment system that accurately predicts the status of a biological sample, will be a valuable tool for deciding which DNA test kit to utilize and how much target DNA to use, when processing compromised forensic samples for DNA testing.

INNULs: A novel design amplification strategy for retrotransposable elements for studying population variation.

OBJECTIVES: Retrotransposable elements (REs), consisting of long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), are a group of markers that can be useful for human identity testing. Until now, however, due to the inherent size difference (up to 6 kb in some instances) associated with insertion and null alleles (or INNULs), the use of REs for facilitated population studies has not been sought or practical. The size of the insertion elements (from a few hundred to several thousand bp) has proven to limit their utility as a marker because of the inefficient amplicon yield with PCR. A novel primer design now facilitates INNUL marker testing. A preliminary panel of single-locus markers was developed to evaluate the potential of typing these insertion elements. Nine INNULs (5 Alu and 4 LINEs) were typed in three major North American populations and analyzed for population genetic features. In addition, the variation of each marker among the sample populations provides insight of its potential use as individual identification or ancestral marker. METHODS: INNUL markers were developed into fluorescently labeled single-loci PCR. Nine markers were developed with amplicons that were less than 180 bp in length, and, depending on the locus amplicons of the INNULs, alleles varied in size from 50 to 1 bp. This allele size is noteworthy because the insertion alleles of the 9 loci range in size from 297 to 6,195 bp. The allele distribution of the INNULs was assessed and analyzed in three major North American populations. RESULTS: Upon observation of the distribution of the alleles in three major North American populations, the markers generally met Hardy-Weinberg expectations, and there was little evidence of detectable levels of linkage disequilibrium. Due to varying distributions of the alleles in the major population groups tested, some of the markers might be better suited for use as an individual identification marker, while others are better suited for bio-ancestral studies. CONCLUSIONS: Using the primer design strategy described in our work, SINEs and (for the first time, to our knowledge) LINEs can be utilized as markers for studying population genetic variation that is more amenable to the limitations of the PCR technique. This study lays the foundation for future work of developing a multiplex panel of INNUL markers that can be used as a single-tube assay for human identity testing utilizing small amplicons (<180 bp), which could be useful for ancient or degraded forensic DNA samples.

Microparticles induce variable levels of activation in macrophages infected with Mycobacterium tuberculosis.

Activation of human macrophages infected with Mycobacterium tuberculosis was investigated following exposure to microparticles (MP) possessing high anti-tubercular efficacy in mice. A small set of innate responses (cytokine profiles, NO production, Annexin-V staining and caspase-8, caspase-9 and caspase-3 activities) of differentiated THP-1 cells or human monocyte-derived macrophages infected 1:10 in vitro were compared. Cytokines of THP-1 macrophages were comparable in trends, but not in magnitude, with five human genotypes studied. MP reversed suppression of tumor necrosis factor induced by infection, and transiently upregulated gamma-interferon. Drug-free MP surprisingly induced gamma-interferon, but not tumor necrosis factor. Primary cells responded to MP, regardless of drug content, by upregulation of NO; but THP-1 cells did not respond to drug-free MP. About 19% of infected cells exposed to MP underwent apoptosis compared to approximately 11% cells treated otherwise. Cell death induced by drug-free MP was caspase independent. Intracellular bacterial survival varied between individuals. Untreated infection resulted in survival of 900+/-141 cfu; exposure to soluble drugs, drug-containing and blank microparticles respectively, reduced CFU counts to <10, <10 and 102+/-139. These observations indicate that despite variations in magnitude between cells from different sources, innate responses conducive to killing intracellular bacteria were evoked by inhalable MP.

Solution-phase synthesis of a library of carbapeptide analogues based on glycosylamino acid scaffolds and their in silico screening and antimicrobial evaluation.

A well-organized and efficient approach toward the solution phase synthesis of a library of carbapeptide analogues based on glycosyl amino ester scaffold is described. The reported synthetic route involves a five step preparation of heptofuranuronamides 6a-h and octopyranuronamide 7e from glycosyl amino esters 1 and 7, respectively. Coupling of glycosyl amino esters 1 or 7 with three different N-Fmoc protected amino acids afford the N-Fmoc protected intermediates 2a-c and 7a. Deprotection of Fmoc group in 2a-c and 7a with piperidine gave respective compounds 3a-c and 7b with free amine. Subsequent coupling of 3a-c and 7b with different aromatic acids furnishes respective heptofuranuronates 4a-h and octopyranuronate 7c in good yields. The latter, on ester hydrolysis by LiOH gave the corresponding glycopeptide analogues 5a-h and 7d with terminal carboxyl group. The carboxyl group in these compounds was amidated with oxalyl chloride/ NH(4)OH to afford heptofuranuronamides 6a-h and octopyranuronamides 7e. In vitro screening of all compounds displayed moderate antifungal, antitubercular, and general antibacterial activities. Reverse docking calculations involving over 841 protein drug targets have identified two potential targets for these compounds. These results will form the basis for synthesizing second-generation antimicrobial compounds.

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.

Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identification.

Human forensic casework requires sensitive quantitation of human nuclear (nDNA), mitochondrial (mtDNA), and male Y-chromosome DNA from complex biomaterials. Although many such systems are commercially available, no system is capable of simultaneously quantifying all three targets in a single reaction. Most available methods either are not multiplex compatible or lack human specificity. Here, we report the development of a comprehensive set of human-specific, target-specific multiplex polymerase chain reaction (PCR) assays for DNA quantitation. Using TaqMan-MGB probes, our duplex qPCR for nDNA/mtDNA had a linear quantitation range of 100 ng to 1 pg, and our triplex qPCR assay for nDNA/mtDNA/male Y DNA had a linear range of 100-0.1 ng. Human specificity was demonstrated by the accurate detection of 0.05 and 5% human DNA from a complex source of starting templates. Target specificity was confirmed by the lack of cross-amplification among targets. A high-throughput alternative for human gender determination was also developed by multiplexing the male Y primer/probe set with an X-chromosome-based system. Background cross-amplification with DNA templates derived from 14 other species was negligible aside from the male Y assay which produced spurious amplifications from other nonhuman primate templates. Mainstream application of these assays will undoubtedly benefit forensic genomics.

Y-chromosome STR system, Y-PLEX 12, for forensic casework: development and validation.

The Y-PLEX 12 system, developed for use in human identification, enables simultaneous amplification of eleven polymorphic short tandem repeat (STR) loci, namely DYS392, DYS390, DYS385 a/b, DYS393, DYS389I, DYS391, DYS389II, DYS 19, DYS439 and DYS438, residing on the Y chromosome and Amelogenin. Amelogenin provides results for gender identification and serves as internal control for PCR. The validation studies were performed according to the DNA Advisory Board's (DAB) Quality Assurance Standards. The minimal sensitivity of the Y-PLEX 12 system was 0.1 ng of male DNA. The mean stutter values ranged between 3.76-15.72%. A full male profile was observed in mixture samples containing 0.5 ng of male DNA and up to 400 ng of female DNA. Amelogenin did not adversely affect the amplification of Y-STRs in mixture samples containing male and female DNA. The primers for the Y-STR loci present in Y-PLEX 12 are specific for human DNA and some higher primates. None of the primate samples tested provided a complete profile at all 11 Y-STR loci amplified with the Y-PLEX 12 system. Y-PLEX 12 is a sensitive, valid, reliable, and robust multiplex system for forensic analysis, and it can be used in human forensic and male lineage identification cases.

Utility of the Y-STR typing systems Y-PLEX 6 and Y-PLEX 5 in forensic casework and 11 Y-STR haplotype database for three major population groups in the United States.

The Y-PLEX 6 and Y-PLEX 5 systems enable analysis for 11 Y-STR loci. We present here the utility of these systems in forensic casework. A total of 188 samples, including 127 evidence samples, were analyzed using either or both of the systems. The evidence sample types included fingernail scrapings, sperm or seminal fluid, epithelial cells, blood and other tissues. The Y-STR typing systems provided useful probative results in difficult cases. A reference database for Caucasian (n = 517), African American (n = 535), and Hispanic (n = 245) population groups within the United States was generated for estimating the haplotype frequency in forensic casework. Among the individuals profiled, 311 Caucasians, 412 African Americans, and 194 Hispanics provided unique profiles in their respective population datasets. This is the first report describing the haplotype database for the set of 11 Y-STR loci recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Linkage analysis reveals that the frequencies from forensically important autosomal loci can be multiplied with the Y-STR haplotype frequency. The results from Y-PLEX systems have been accepted in courts in the United States.

Quantitative PCR for DNA identification based on genome-specific interspersed repetitive elements.

We have designed and evaluated a series of class-specific (Aves), order-specific (Rodentia), and species-specific (equine, canine, feline, rat, hamster, guinea pig, and rabbit) polymerase chain reaction (PCR)-based assays for the identification and quantitation of DNA using amplification of genome-specific short and long interspersed elements. Using SYBR Green-based detection, the minimum effective quantitation levels of the assays ranged from 0.1 ng to 0.1 pg of starting DNA template. Background cross-amplification with DNA templates derived from sixteen other species was negligible prior to 30 cycles of PCR. The species-specificity of the PCR amplicons was further demonstrated by the ability of the assays to accurately detect known quantities of species-specific DNA from mixed (complex) sources. The 10 assays reported here will help facilitate the sensitive detection and quantitation of common domestic animal and bird species DNA from complex biomaterials.

Development and validation of the Y-PLEX 5, a Y-chromosome STR genotyping system, for forensic casework.

A genotyping system, Y-PLEX 5, has been developed for use in human identification. The Y-PLEX 5 enables simultaneous amplification of five polymorphic short tandem repeat (STR) loci residing on the Y-chromosome, which are DYS389I, DYS389II, DYS439, DYS438, and DYS392. As little as 0.1 ng of template DNA can be used for analysis. The specificity of the amplification reaction enabled analysis of male DNA in a male: female DNA mixture at a ratio of 1:600. Mean stutter values ranged from 3.60-10.97%. Among the different primates investigated, the DNA from orangutan exhibited amplification at DYS438 locus and from gorilla at DYS439 and DYS438 loci. The DNA from cat, dog, and horse did not yield any amplified product. Studies on development of the genotyping system, generation and description of the allelic ladder, and validation of the multiplex PCR as per the FBI Director's Quality Assurance Standards were carried out. Y-STR allele and haplotype frequencies in two populations were generated. The data indicate that the Y-PLEX 5 genotyping system is sensitive and reliable, and can be used in human forensic and male lineage identification cases.

Human DNA quantitation using Alu element-based polymerase chain reaction.

Human forensic casework requires sensitive quantitation of human nuclear DNA from complex sources. Widely used commercially available systems detect both nonhuman and human primate DNA, often require special equipment, and have a detection limit of approximately 0.1ng. Multicopy Alu elements include recently integrated subfamilies that are present in the human genome but are largely absent from nonhuman primates. Here, we present two Alu element-based alternative methods for the rapid identification and quantitation of human DNA, inter-Alu PCR and intra-Alu PCR. Using SYBR green-based detection, the effective minimum threshold level for human DNA quantitation was 0.01ng using inter-Alu- and 0.001ng using intra-Alu-based PCR. Background cross-amplification with nonhuman DNA templates was detected at low levels using inter-Alu-based PCR, but was negligible using intra-Alu-based PCR. These Alu-based methods have several advantages over currently available systems. First, the assays are PCR based and no additional unique equipment is required. Second, the high copy number of subfamily-specific Alu repeats in the human genome makes these assays human specific within a very sensitive linear range. The introduction of these assays to forensic laboratories will undoubtedly increase the sensitivity and specificity of human DNA detection and quantitation from complex sources.

Quantitative intra-short interspersed element PCR for species-specific DNA identification.

We have designed and evaluated four assays based upon PCR amplification of short interspersed elements (SINEs) for species-specific detection and quantitation of bovine, porcine, chicken, and ruminant DNA. The need for these types of approaches has increased drastically in response to the bovine spongiform encephalopathy epidemic. Using SYBR Green-based detection, the minimum effective quantitation levels were 0.1, 0.01, 5, and 1 pg of starting DNA template using our bovine, porcine, chicken, and ruminant species-specific SINE-based PCR assays, respectively. Background cross-amplification with DNA templates derived from 14 other species was negligible. Species specificity of the PCR amplicons was further demonstrated by the ability of the assays to accurately detect trace quantities of species-specific DNA from mixed (complex) sources. Bovine DNA was detected at 0.005% (0.5 pg), porcine DNA was detected at 0.0005% (0.05 pg), and chicken DNA was detected at 0.05% (5 pg) in a 10-ng mixture of bovine, porcine, and chicken DNA templates. We also tested six commercially purchased meat products using these assays. The SINE-based PCR methods we report here are species-specific, are highly sensitive, and will improve the detection limits for DNA sequences derived from these species.

Development and validation of a multiplexed Y-chromosome STR genotyping system, Y-PLEX 6, for forensic casework.

A Y-chromosome multiplex polymerase chain reaction (PCR) amplification kit, known as Y-PLEX 6, has been developed for use in human identification. The Y-PLEX 6 kit enables simultaneous amplification of six polymorphic short tandem repeat (STR) loci located on the non-recombinant region of the human Y-chromosome. These loci are: DYS393, DYS19, DYS38911, DYS390, DYS391, and DYS385. Our studies show that as little as 0.2 ng of template DNA can be used for analysis. The specificity of the amplification reaction enabled analysis of male DNA in a male:female DNA mixture at a ratio of 1:125. Among the six Y-STR loci, the maximum mean stutter percentage was 11.9 for allele at DYS38911 locus. Attempts at amplification of DNA from various animal sources revealed that the Y-PLEX 6 primers are human specific. Details of the development of the kit, generation and description of the allelic ladders, and validation of the multiplex PCR are presented. In addition, Y-STR allele and haplotype frequencies in three populations have been investigated. The data indicate that results obtained using the Y-PLEX 6 kit are robust, sensitive, and reliable and can be used in human forensic and male lineage identification cases.

DNA profiling of azoospermic semen samples from vasectomized males by using Y-PLEX 6 amplification kit.

Post-vasectomized azoospermic semen samples (N = 6) were analyzed for short tandem repeats (STR) on the Y-chromosome by using Y-PLEX 6 and the 310 Genetic Analyzer. We have observed a wide variation in the yield of extracted DNA from 12.5-1,000 ng. This variation was attributed to the number of epithelial and/or white blood cells that are present in these azoospermic samples. DNA profiles of these vasectomized males were obtained for all six Y-STR loci, namely DYS393, DYS 19, DYS389II, DYS390, DYS391, and DYS385 amplified by using the Y-PLEX 6.