Can brothers share the same STR profile?

This report demonstrates the limits of DNA identification when siblings are involved.The Israeli DNA database routinely amplifies suspects samples using the PowerPlex^® ESI16 system (Promega). While uploading a series of suspects into the database software, we found an unusual high number of shared alleles between two suspects 31 out of 32 alleles. Verification of their demographic data identified them as brothers. After confirmation of their paternity affiliation using the AmpFlSTR^®YFiler™ (Applied Biosystems), we used two other multiplexes kits to improve the differentiation rate. The PowerPlex^® ESX17 System (Promega) added one locus, SE33, who exhibits four different alleles. The second kit, the AmpFlSTR^®MiniFiler™ (Applied Biosystems) added three more loci. Only one allele difference was found.In order to increase the discrimination power between related and unrelated individuals, we recommend that the DNA laboratories consider using a larger multiplex typing kit in cases like the one informed here.     

Null alleles and sequence variations at primer binding sites of STR loci within multiplex typing systems

Rare variants are widely observed in human genome and sequence variations at primer binding sites might impair the process of PCR amplification resulting in dropouts of alleles, named as null alleles. In this study, 5 cases from routine paternity testing using PowerPlex^®21 System for STR genotyping were considered to harbor null alleles at TH01, FGA, D5S818, D8S1179, and D16S539, respectively. The dropout of alleles was confirmed by using alternative commercial kits AGCU Expressmarker 22 PCR amplification kit and AmpFℓSTR^®. Identifiler^® Plus Kit, and sequencing results revealed a single base variation at the primer binding site of each STR locus. Results from the collection of previous reports show that null alleles at D5S818 were frequently observed in population detected by two PowerPlex^® typing systems and null alleles at D19S433 were mostly observed in Japanese population detected by two AmpFℓSTR™ typing systems. Furthermore, the most popular mutation type appeared the transition from C to T with G to A, which might have a potential relationship with DNA methylation. Altogether, these results can provide helpful information in forensic practice to the elimination of genotyping discrepancy and the development of primer sets.     

Validation and assessment of the Investigator® 24plex QS kit for forensic casework application: Comparison with the PowerPlex® fusion system and GlobalFiler™ PCR amplification kits

Casework evidence samples are likely to be placed under diverse and harsh environments as compared to quantified DNA samples including serial-diluted standard DNA samples. Internal validation of a novel STR kit using casework evidence sample, which is conducted according to various conditions such as DNA contamination and degradation, is crucial before being used as a forensic application. Therefore, this study aimed to elucidate the reliability of the Investigator® 24plex QS kit through DNA derived from casework evidence and to assess whether it is applicable to STR analysis together with PowerPlex® Fusion System and GlobalFiler™ PCR Amplification Kit. DNA was extracted from 189 casework evidence samples in a total of 77 cases. The mismatch of the allelic size of this kit through allelic sizing precision test, was suitable according to ENFSI guidelines. All heterozygous balance of the three kits were above 0.6 recommended value of ENFSI guideline. The number of allele drop-in was most frequent in the GlobalFiler™ PCR Amplification Kit. In addition, the number of allele drop-out was most frequent in the Investigator® 24plex QS kit. The cutoff concentration of DNA detected in three kits of one complete STR was approximately 45 pg/μL on average. Despite of several limitations, the Investigator® 24plex QS kit is considered to have the capability to be used for STR analysis of casework evidence samples.     

Recognizing the advantage of employing the PowerPlex® ESI and PowerPlex® ESX complementary kits for purposes of clarification in routine casework

In an effort to promote European cross-border cooperation in fighting crime and international terrorism the Treaty of Prüm was drafted and accepted within the European forensic community. This move led to the commercial development of new multiplex kits which introduced five new STR loci and promised better performance. Recently the Israel Police DNA Casework and Database laboratories adopted the PowerPlex^® ESI kit for routine use in our laboratories. Presented in this paper are examples of three types of ambiguous results encountered during the implementation of the PowerPlex^® ESI kit into routine work. These ambiguous products presented themselves in the form of (1) extreme variants outside of loci borders, (2) failure to amplify sister allele pairs or expression of null alleles and (3) episodes of loss of separation of adjacent microvariants primarily in mixture samples. The re-analysis of all these samples using the PowerPlex^® ESX kit successfully and rapidly clarified all three categories of anomalies.Spotlighting such events to the forensic community, especially regarding the novel loci introduced in these next generation kits, can aid in raising the analyst's awareness to their future appearances and prevent possible erroneous conclusions. In addition, providing timely DNA results to investigating teams is of great importance and operational forensic laboratories do not have at their immediate disposal methods such as sequencing to elucidate such manifestations. We suggest that the complementary use of the PowerPlex^® ESI and PowerPlex^® ESX can provide a benefit for clarification purposes in routine casework.     

Comparative study of STR loci for typing old skeletal remains with modified protocols of AmpFlSTR Identifiler and AmpFlSTR MiniFiler STR Kits

This study highlights a comparative study of short tandem repeat (STR) loci with modified protocols of AmpFlSTR Identifiler and AmpFlSTR MiniFiler STR Kits for typing 27 old skeletal remains collected from 100–1000-year-old mass graves in Pakistan. DNA profiles were obtained from minute quantities of DNA (even from?≤?10?pg/μL) with modified protocols of these kits, which is a significant achievement in this study. Consensus profiles were produced for each bone sample. A comparison was carried out between Identifiler and Minifiler successfully genotyped STR loci. Full concordance was perceived in 97.33% (146/150) of the compared STR loci, while discordant STR loci were 2.67% (4/150) of the total successfully genotyped STR loci due to either or both allele drop-out or drop-in. Finally, it was observed that the AmpFlSTR MiniFiler kit promoted the recovery of locus/alleles that failed to type with the AmpFlSTR Identifiler kit and more informative DNA profiles were obtained from old skeletal remains with the AmpFlSTR MiniFiler STR kit compared with the AmpFlSTR Identifiler STR kit.     

Developmental validation of the AmpFℓSTR® NGM SElect™ PCR Amplification Kit: A next-generation STR multiplex with the SE33 locus

The AmpFℓSTR^® NGM SElect™ PCR Amplification Kit is a new 17-plex STR genotyping kit designed for use primarily in forensic casework analysis. The kit was designed to be a counterpart to the AmpFℓSTR^® NGM™ Kit for laboratories wishing to add the SE33 locus to the new European Standard Set of STR loci. The NGM SElect Kit shares the same primer sets for 16 common loci with the NGM Kit (D10S1248, D3S1358, vWA, D16S539, D2S1338, amelogenin, D8S1179, D21S11, D18S51, D19S433, TH01, FGA, D22S1045, D2S441, D1S1656 and D12S391), with additional primers for the SE33 locus. Developmental validation studies followed the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines for STR kit manufacturers and tested several critical areas of kit performance including a sensitivity series, DNA mixtures and inhibited samples. The studies demonstrated that the NGM SElect Kit provides equivalent overall performance to the NGM Kit, but with even greater discriminatory power due to the inclusion of the highly informative SE33 locus.     

A review of DNA profiling success for laser microdissected forensic casework samples

ABSTRACT

Laser microdissection (LMD) is a tool that is used in forensic laboratories for the analysis of DNA from specifically targeted cells. Since 2010, the Institute of Environmental Science and Research Limited’s (ESR) Forensic Biology laboratory has applied LMD DNA testing to a variety of sexual assault casework samples where small numbers of sperm are present in cell mixtures. In this paper we review the DNA profiling results obtained from semen-stained casework samples that have been analysed using the LMD DNA methodology developed in our laboratory. Dissected sperm have been analysed using the AmpFISTR Identifiler amplification kit at 28 cycle PCR, the AmpFISTR MiniFiler amplification kit at 30 cycle PCR, or the AmpFISTR SGM Plus amplification kit at 34 cycle PCR, depending on the number of sperm recovered and on consideration of other circumstantial case information, such as the time since intercourse (TSI). From a review of these data, success rates for different sample numbers of sperm recovered from semen-stained samples are determined. The DNA profiling results obtained from three cases where laser microdissection has been used are also presented to demonstrate the success of the LMD testing strategy in a forensic laboratory.     

Korean population genetic data and concordance for the PowerPlex® ESX 17, AmpFlSTR Identifiler®, and PowerPlex® 16 systems

In case of paternity or maternity investigations with short tandem repeat (STR) analysis, deficient cases, missing person, or mutations are encountered and common STRs cannot provide sufficient forensic parameters. Thus, it is recommended that additional STRs are needed to complement conventional analysis for more reliable forensic information. We analyzed variation of 23 STRs contained in the new PowerPlex^® ESX 17 kit (Promega) and two conventional kits of the AmpFlSTR Identifiler^® (Applied Biosystems) and PowerPlex^® 16 systems (Promega) in 452 randomly chosen individuals from Korea to provide an expanded and reliable Korean database. Allele frequencies and forensic parameters were used to evaluate suitability and robustness of the new kit for forensic genetic analysis as well as in concordance studies. The combined probability of match for the 16 loci in the PowerPlex^® ESX 17 system was 2.76 × 10⁻²⁰. One genotyping discrepancy due to a null allele was observed at the D18S51 locus (the concordant rate = 99.99%), showing a primer-binding site mutation in the sequence of the locus (G-to-A substitution at position 146 of Genbank accession number JX018211). Thus, the new kit is a valuable forensic tool and is suitable to extend the Korean population genetic data obtained with well-established polymerase chain reaction multiplex-kits of the AmpFlSTR Identifiler^® and PowerPlex^® 16 systems.     

The Qiagen Investigator® Quantiplex HYres as an alternative kit for DNA quantification

The Investigator^® Quantiplex HYres kit was evaluated as a potential replacement for dual DNA quantification of casework samples. This kit was determined to be highly sensitive with a limit of quantification and limit of detection of 0.0049 ng/μL and 0.0003 ng/μL, respectively, for both human and male DNA, using full or half reaction volumes. It was also accurate in assessing the amount of male DNA present in 96 mock and actual casework male:female mixtures (various ratios) processed in this exercise. The close correlation between the male/human DNA ratios expressed in percentages derived from the Investigator^® Quantiplex HYres quantification results and the male DNA proportion calculated in mixed AmpFlSTR^® Profiler^® Plus or AmpFlSTR^® Identifiler^® Plus profiles, using the Amelogenin Y peak and STR loci, allowed guidelines to be developed to facilitate decisions regarding when to submit samples to Y-STR rather than autosomal STR profiling. The internal control (IC) target was shown to be more sensitive to inhibitors compared to the human and male DNA targets included in the Investigator^® Quantiplex HYres kit serving as a good quality assessor of DNA extracts. The new kit met our criteria of enhanced sensitivity, accuracy, consistency, reliability and robustness for casework DNA quantification.     

Evaluation of the RapidHIT™ 200 System: A comparative study of its performance with Maxwell® DNA IQ™/Identifiler® Plus/ABI 3500xL workflow

RapidHIT™ System is a rapid DNA instrument that is capable of processing forensic samples from extraction through to capillary electrophoresis and profile generation within two hours. Evaluation of the RapidHIT™ 200 System was conducted to examine several key performance indicators of the instrument, including reproducibility, contamination, sensitivity, versatility and the possibility of sample re-extraction. Results indicated that the RapidHIT™ 200 System was capable of generating high quality DNA profiles which were comparable to those from the standard protocol comprising of Maxwell^® 16 DNA IQ™ System, Identifiler^® Plus and ABI 3500xL. No contamination was detected during the studies. Results also showed that the instrument was able to generate DNA profiles from samples containing lower amounts of DNA (0.5 μl of blood) albeit with more allele and locus dropouts when compared to the standard protocol. The ability to process blood swabs, blood-stained FTA punches, semen swabs, buccal swabs, product of conception (POC), bone marrow, fingernail clippings and cigarette butts at a good success rate indicated the robustness and versatility of the RapidHIT™ 200 System. Furthermore, additional alleles could be recovered via re-analysis of the failed samples using the standard protocol. In summary, our results showed that the RapidHIT™ 200 System was able to process casework samples for the purpose of providing rapid intelligence through DNA database searches and reference matching. Confirmative DNA results can be obtained through either concurrent processing of duplicate samples via standard protocol or re-extraction of samples retrieved from the RapidHIT™ sample cartridge.     

Prototype PowerPlex® Y23 System: A concordance study

The Prototype PowerPlex^® Y23 System (Promega Corporation, Madison, WI) is a polymerase chain reaction-based amplification kit that targets the 23 Y STR loci DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, and Y-GATA-H4. A total of 951 samples from six populations were typed to evaluate the kit and examine concordance for 17 of the loci that are in common with those that can be typed using the AmpFlSTR^® Yfiler™ kit (Life Technologies, Carlsbad, CA). A total of 16,167 loci were analyzed for each multiplex, and overall concordance was observed. Because of different kit designs, and although concordant for the genetic type, discordant calls can occur due to a deletion at the DYS448 locus. Users should take into consideration such nomenclature anomalies when comparing Y STR profiles. This new kit allows a large battery of Y STR loci to be analyzed using the same basic technologies already employed in forensic laboratories.     

Variants observed for STR locus SE33: a concordance study

Discordance of STR typing results can be expected between kits that employ different primers for amplification. The complex motif of the SE33 locus and its flanking regions can contribute to the degree of discordant results. Sequence-dependent conformational changes can manifest as length differences under certain electrophoretic conditions and/or use of different primers. The AmpFlSTR^® NGM SElect? PCR Amplification Kit (Life Technologies, Carlsbad, CA), PowerPlex^® ESX 17 system (Promega Corporation, Madison, WI), and PowerPlex^® ESI 17 system (Promega Corporation) were compared for concordance of allele calls for the SE33 marker in selected samples. A total of 16 samples were identified that were discordant at one of the SE33 alleles by an apparent one nucleotide in size. While the ESX 17 and NGM SElect? kits yielded concordant results for these 16 samples, the ESI 17 kit generated alleles that differed. The discordant alleles were observed in individuals of African and European descent. Sequence analysis revealed that the one-base difference in size is not due to an indel but is instead the result of a single nucleotide polymorphism (SNP) in the flanking region of the SE33 repeat region. Three different SNPs were observed, one of which is novel.Although these migration anomalies were observed only with the ESI 17 kit, one cannot preclude that a similar phenomenon may occur with the other kits as data sets increase. The type and degree of discordance of STR allele calls among STR kits is an important issue when comparing STR profiles among laboratories and when determining search parameters for identifying candidate associations in national databases.     

A comparison of four methods for PCR inhibitor removal

Biological samples collected from the crime scenes often contain some compounds that can inhibit the polymerase chain reaction (PCR). The removal of PCR inhibitors from the extracts prior to the PCR amplification is vital for successful forensic DNA typing. This paper aimed to evaluate the ability of four different methods (PowerClean^® DNA Clean-Up kit, DNA IQ™ System, Phenol–Chloroform extraction and Chelex^®-100 methods) to remove eight commonly encountered PCR inhibitors including: melanin, humic acid, collagen, bile salt, hematin, calcium ions, indigo and urea. Each of these PCR inhibitors was effectively removed by the PowerClean^® DNA Clean-Up kit and DNA IQ™ System as demonstrated by generating more complete short tandem repeat (STR) profiles from the cleaned up inhibitor samples than from the raw inhibitor samples. The Phenol–Chloroform extraction and Chelex^®-100 methods, however, could only remove some of eight PCR inhibitors. Our results demonstrated that the PowerClean^® DNA Clean-Up kit and DNA IQ™ System were very effective for the removal of known PCR inhibitors that are routinely found in DNA extracts from forensic samples.     

Developmental validation of the PowerPlex® ESI 16/17 Fast and PowerPlex® ESX 16/17 Fast Systems

The PowerPlex^® ESI 16 Fast, ESI 17 Fast, ESX 16 Fast, and ESX 17 Fast Systems represent faster cycling versions (50 min or less) of the PowerPlex^® ESI and ESX Systems released by Promega in 2009 to accommodate the ENFSI and EDNAP groups’ call for new STR multiplexes for Europe. In addition to amplification of purified DNA samples, these new faster cycling systems allow for direct amplification from single-source blood and buccal samples deposited on FTA^® and nonFTA paper as well as from SwabSolution™ extracts of buccal swabs without the need for purification and quantitation. There are no changes to the autosomal primer pair sequences in the PowerPlex^® ESI Fast and ESX Fast Systems compared to the original multiplexes, and full concordance at all autosomal loci and amelogenin was observed with data generated previously with the original PowerPlex^® ESI and ESX Systems. This paper describes the developmental validation study performed on these new fast systems following guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and those of the DNA Advisory Board (DAB). Validation data demonstrate that these systems are sensitive for detecting low levels of DNA while also being capable of generating robust profiles from the high amount of input DNA present in direct-amplification samples. These systems are also tolerant to both high concentrations of PCR inhibitors as well as to slight variations in the final concentration of master mix and primer pair present in the amplification reaction that might be encountered due to pipetting error. The results of this validation study demonstrate that these systems may be used on multiple thermal cyclers and capillary electrophoresis platforms.     

Comparison of different interpretation strategies for low template DNA mixtures

DNA typing protocols have been improved over the last few years and even mixtures from minute and low grade DNA stains do not necessarily preclude typing. Nevertheless, in those cases stochastic phenomena tend to hamper interpretation. In order to supplement the current discussion about the interpretation of such challenging data, we focused on different combinations of analyses as an attempt to overcome stochastic problems.We analyzed mixtures of two types of degraded DNA in low template amounts (50 and 100 pg DNA per contributor) using four types of multiplex STR typing kits: PowerPlex^® ESX 17, PowerPlex^® ESI 17, Investigator^® ESSplex SE Kit and P11, a non-commercial kit. We employed the results of double or triple analyses for a comparison of different types of interpretation rules based on reporting either only reproducible alleles (consensus interpretation) or all alleles, even if they are only observed once (composite interpretation). The interpreted and composed profiles were compared to the known alleles of the contributors and a “degree of validity” was calculated.When only two single amplifications were taken into account, we observed a higher degree of validity for composite profiles. The difference for consensus interpretation could be compensated when a minimum of three amplifications were carried out.Using the same kit for repeat analyses increases the chances to yield reproducible results required for consensus interpretation. Combining different kits in a complementing approach, on the other hand, offers the opportunity to reduce the number of drop-out alleles: differences in amplicon lengths for specific markers between kits can increase the resulting information. In the case of a few amplifications available this effect might only be visible with the composite method. Several markers like SE33 are particularly affected by this.Based on our observations the consensus interpretation method may not reflect the original profile in an optimal way in some special cases like low template, degraded mixture stains. In those cases the composite interpretation could yield more complete results. However, such a composite profile should be used with caution and only for limited purposes. Generally recommending the consensus interpretation thus seems not to be justified: a more differentiated approach appears to be worthwhile, e.g. the amount of drop-outs, the number of replicates, choice and combination of kits and even a marker specific procedure might be taken into account.     

Assessing PreCR™ repair enzymes for restoration of STR profiles from artificially degraded DNA for human identification

Forensic scientists have used several approaches to obtain short tandem repeat (STR) profiles from compromised DNA samples, including supplementing the polymerase chain reaction (PCR) with enhancers and using procedures yielding reduced-length amplicons. For degraded DNA, the peak intensities of the alleles separated by electrophoresis generally decrease as the length of the allele increases. When the intensities of the alleles decrease below an established threshold, they are described as drop-outs, thus contributing to a partial STR profile. This work assesses the use of repair enzymes to improve the STR profiles from artificially degraded DNA. The commercial PreCR™ repair kit of DNA repair enzymes was tested on both purified DNA and native DNA in body fluids exposed to oxidizing agents, hydrolytic conditions, ultraviolet (UV) and ionizing radiation, and desiccation. The strategy was to restrict the level of DNA damage to that which yields partial STR profiles in order to test for allele restoration as opposed to simple allele enhancement. Two protocols were investigated for allele restoration: a sequential protocol using the manufacturer's repair procedure and a modified protocol reportedly designed for optimal STR analysis of forensic samples. Allele restoration was obtained with both protocols, but the peak height appeared to be higher for the modified protocol (determined by Mann–Kendall Trend Test). The success of the approach using the PreCR™ repair enzymes was sporadic; it led to allele restoration as well as allele drop-out. Additionally, allele restoration with the PreCR™ enzymes was compared with restoration by alternative, but commonly implemented approaches using Restorase™, PCRBoost™, bovine serum albumin (BSA) and the Minifiler™ STR system. The alternative methods were also successful in improving the STR profile, but their success also depended on the quality of the template encountered. Our results indicate the PreCR™ repair kit may be useful for restoring STR profiles from damaged DNA, but further work is required to develop a generalized approach.     

Direct amplification of casework bloodstains using the Promega PowerPlex® 21 PCR Amplification System

A significant number of evidence items submitted to Forensic Science Service Tasmania (FSST) are blood swabs or bloodstained items. Samples from these items routinely undergo phenol:chloroform:isoamyl alcohol organic extraction and quantitative Polymerase Chain Reaction (qPCR) testing prior to PowerPlex^® 21 amplification. This multi-step process has significant cost and timeframe implications in a fiscal climate of tightening government budgets, pressure towards improved operating efficiencies, and an increasing emphasis on rapid techniques better supporting intelligence-led policing.Direct amplification of blood and buccal cells on cloth and Whatman FTA™ card with PowerPlex^® 21 has already been successfully implemented for reference samples, eliminating the requirement for sample pre-treatment. Scope for expanding this method to include less pristine casework blood swabs and samples from bloodstained items was explored in an endeavour to eliminate lengthy DNA extraction, purification and qPCR steps for a wider subset of samples.Blood was deposited onto a range of substrates including those historically found to inhibit STR amplification. Samples were collected with micro-punch, micro-swab, or both. The potential for further fiscal savings via reduced volume amplifications was assessed by amplifying all samples at full and reduced volume (25 and 13 μL).Overall success rate data showed 80% of samples yielded a complete profile at reduced volume, compared to 78% at full volume. Particularly high success rates were observed for the blood on fabric/textile category with 100% of micro-punch samples yielding complete profiles at reduced volume and 85% at full volume.Following the success of this trial, direct amplification of suitable casework blood samples has been implemented at reduced volume. Significant benefits have been experienced, most noticeably where results from crucial items have been provided to police investigators prior to interview of suspects, and a coronial identification has been successfully completed in a short timeframe to avoid delay in the release of human remains to family members.     

Improved STR profiles from improvised explosive device (IED): fluorescence latent DNA detection and direct PCR

Bombing accounts for the largest share of terrorist incidents worldwide. Most involve an improvised explosive device (IED): a bomb made from household items. Touch DNA may be left on parts of an IED during assembly. However, an IED conflagration degrades DNA, and there has never been a way to locate where touch DNA may remain. To solve this problem, we combined the use of fluorescent dye to locate latent DNA and direct PCR to improve STR profiles of DNA obtained from IEDs. Six fluorescent DNA-binding dyes were evaluated at various concentrations for the purpose of staining latent DNA. SYBR® Green I and Diamond™ Nucleic Acid dye were able to visualize touch DNA on IED substrates. Inhibition studies with extracted DNA and touch DNA using both dyes revealed that Diamond™ dye inhibited direct STR amplification, while SYBR® Green I did not. Stability studies at three temperatures showed optimum performance of SYBR® Green I up to 24 h after formulation. As such, only SYBR® Green I was further used to develop a “visualized-direct PCR” method. Using the conventional approach and the novel “visualized-direct PCR” approach in a single-blind investigation of mock IED evidence, the “visualized-direct PCR” approach had a 98.6% chance of obtaining more alleles (95% highest density interval (HDI): 0.7 to 10.0 alleles). A decrease in non-donor’s alleles (mixed profiles) was also observed. The developed approach has the potential to revolutionize the process of STR typing from touch DNA.     

Assessment of application value of 19 autosomal short tandem repeat loci of GoldenEyeTM 20A kit in forensic paternity testing

This study was carried out to assess the application value of 19 autosomal short tandem repeat (STR) loci of GoldenEye^TM 20A kit, in which 13 combined DNA index system core STR loci and PentaE, PentaD, D2S1338, D19S433, D12S391, and D6S1043 of six STR loci could be used in forensic paternity testing in Chinese population. We amplified the genomic DNA from blood samples on FTA paper of 289 paternity testing cases by using the GoldenEye^TM 20A kit. The amplified products were detected by capillary electrophoresis, and then the genotypes of 20 genetic markers including 19 STR loci as well as Amelogenin for sex determination were analyzed by GeneMapper v3.2 and GeneMarker HID Software. The results of genotypes were compared to the three commonly used commercial kits including AmpF?STR Identifiler^TM, PowerPlex^TM16, and AmpF?STR Sinofiler^TM kits. Compared to the three other common commercial kits, the GoldenEye^TM 20A kit had higher value of combined paternity index in certainty of paternity or non-exclusion paternity cases, and more numbers of STR loci were excluded in exclusionary paternity cases. Our data in this study showed that the GoldenEye^TM 20A kit has a higher application value in forensic paternity testing and will be of help for kinship analysis.     

Developmental validation of a fully integrated sample-to-profile rapid human identification system for processing single-source reference buccal samples

Short tandem repeat (STR) DNA typing is a global standard for human identification. Current practice involves highly trained forensic analysts, operating in a laboratory setting, using multiple instruments to process samples and analyze the data. Here, we report the developmental validation of a fully integrated and automated DNA profiling system, the RapidHIT^® System, capable of producing up to five high quality STR profiles with full controls in approximately 90 min using PowerPlex^®16 HS RapidHIT chemistry. The system integrates all sample handling steps: starting from lysis of cells on buccal swabs or other buccal sample types through DNA extraction, normalization, amplification,capillary array electrophoresis, detection, and integrated software analysis.The results describe the developmental validation of the RapidHIT™ System for buccal samples processed with the DNA IQ™ extraction chemistry using a guandinium chaotropic agent and paramagnetic beads followed by amplification using a modified version of PowerPlex 16 HS chemistry (PowerPlex 16 HS RapidHIT chemistry), and capillary electrophoresis with manual review of genotyping data following interpretation guidelines. All processing from the buccal swab to generation and processing of the profile occurs on the RapidHIT platform.Resultare concordant with traditional methods, with 88% first pass success rates for both the CODIS and PowerPlex 16 loci. Average peak height ratios were 0.89 for buccal swabs. The system produces full profiles from swabs with at least 176 ng of saliva DNA. Rapid DNA identification systems will significantly enhance capabilities for forensic labs, intelligence, defense, law enforcement, refugee and immigration applications, and kinship analysis.