Multiplex Short Tandem Repeat Research Articles

English

Short tandem repeat (STR) markers are extensively being used for human identification as well as paternity and forensic case work. X-Chromosome STR (X-STR) markers are a powerful complementary system especially in deficiency paternity testing. This study presents development of a new X-chromosomal STR multiplex kit providing short-amplicon (<200 bp) fragments. About 200 samples from Punjabi population were typed for seven X-STR markers: DXS101, DXS6789, DXS7132, DXS7423, DXS8378, GATA172D05 and GATA31E08. A total of 51 alleles were detected in the range of five to 10 alleles for each marker. The data can be used as reference database for Punjabi population along with the current battery of autosomal STR for forensic case work to increase the discrimination capacity and strengthen the existing system.   Key words: X-chromosome short tandem repeats (X-STRs), mini-STRs, Punjabi population.

Paternity testing and forensic DNA typing by multiplex STR analysis using ABI PRISM 310 Genetic Analyzer

Abstract Short tandem repeats (STRs) are widespread throughout the human genome and are a rich source of highly polymorphic markers which can be detected by PCR. To gain a better appreciation for how the polymorphism at a particular locus impacts the individual identity, the present study was undertaken to explore the use of 15 STR loci in forensic investigation and paternity testing. Multiplex STR typing was used to study the 15 STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA) in addition to a gender identification marker, amelogenin, by capillary electrophoresis on 310 Genetic Analyzer. Samples from 85 trio and duo cases of disputed paternity were investigated. The data were analyzed to give information on paternity index, probability of paternity, frequency of number of exclusions and rate of mismatch at each STR locus. The method was also successfully applied to forensic personal identification in theft and murder cases. The results demonstrated that the STR typing is a reliable and robust tool for analyzing the forensic practice as well as for paternity testing. The advantages of using multiplex STR analysis over other conventional methods are discussed.

Evaluation of the sensitivity of two recently developed STR multiplexes for the analysis of chimerism after haematopoietic stem cell transplantation

Forensic-oriented kits analysing short tandem repeat (STR) polymorphisms are widely used to determine the proportions of donor and recipient cells after haematopoietic stem cell transplantation. The sensitivity of this technology is crucial for the early detection of relapse and, in consequence, the adjustment of the treatment to enhance donor-origin haematopoiesis in transplant recipients. The objective of this study was to compare the performance of two recently developed STR multiplex kits, AmpFℓSTR(®) Identifiler(®) Plus PCR Amplification Kit (Applied Biosystems) and Investigator™ IDplex(®) (Qiagen), in the analysis of chimerism. Fifteen STR loci were amplified with both kits in 26 peripheral blood samples of transplantated patients showing chimerism. Peak amplitude threshold, detection limit (%DL), per cent donor chimerism and efficacy of each multiplex and STR were determined, and the results with both kits were compared. The %DL and the estimated per cent donor chimerism were similar with both kits. On the other hand, Identifiler(®) Plus kit allowed chimerism identification only in 24 (92%) of the 26 cases with chimerism detected by using the Investigator™ IDplex(®) when only 'type 5' allelic constellations (i.e. without potential interference by stutter peaks) were taken into account. However, IDplex(®) efficacy was somewhat lower than that of Identifiler Plus when only the most informative loci (D2S1338, D21S11, D18S51 and FGA) were considered. Therefore, although each system had some particular advantages and disadvantages, overall both STR multiplexes showed similar performance in qualitative and quantitative chimerism analysis.

The validation of a 15 STR multiplex PCR for Cannabis species

Trade and acquisition of Cannabis drugs are illegal in many countries worldwide; nevertheless, crimes related with these drugs are a major problem for the investigative authorities. With this manuscript, we want to introduce a 15 short tandem repeat (STR) Cannabis marker set that can be amplified in one PCR reaction. This multiplex PCR is specific to Cannabis species and combines highly informative STR markers. The 15 STR multiplex is easy to use and was validated according to common laboratory quality standards. Due to the fact that a lot of Cannabis plants are cultivated by clonal propagation and may show aneuploidy, polyploidy or multiple gene loci, it is not possible to apply biostatistics that follow the Hardy-Weinberg law. However, this multiplex will help the police to trace back trade routes of drug syndicates or dealers and it can help to link Cannabis plants to a crime scene.

Comparison of two STR multiplexes for the analysis of chimerism after hematopoietic stem-cell transplantation

Short-tandem repeat (STR) genotyping is widely used for forensic purposes and it is also used to determine the proportions of donor and recipient cells after hematopoietic stem-cell transplantation. New STR multiplexes have recently been developed to analyze the complete CODIS plus D19S433 and D2S1338, such as AmpFℓSTR ® Identifiler ® Plus PCR Amplification kit (Applied Biosystems) and Investigator™ IDplex ® (Qiagen). The objective of this study was to compare the sensitivity of both kits in the analysis of chimerism. The Investigator™ IDplex ® allowed quimerism detection only in 10 (67%) of the 15 cases with chimerism detected by using the Identifiler ® Plus kit, suggesting that the latter may have higher sensitivity.

Comparison of Identifiler ®, Identifiler Plus ® and Minifiler ® performance in an initial paternity testing study on old skeletal remains at the forensic and legal medicine area of the Government of Andorra (Pyrenees)

The problem oftentimes faced by forensic scientists when working with DNA extracted from exhumations and skeletal remains is either DNA degradation or DNA contamination. Various methods have been used to improve the identification of skeletal remains using DNA technology. Most of these systems include short tandem repeat (STR) analyses. Herein, we describe our work on the identification of individuals involved in a paternity test where all parents, the mother and the two alleged fathers, had died between 10 and 58 years ago. Trace amounts of highly degraded human DNA were successfully extracted from each bone. Despite the presence of DNA inhibitors in the amplification, microsatellite alleles could be reproducibly amplified from the femur. DNA was extracted from bone which was turned into fine powder, then proteolytic digestion followed by phenol/chloroform purification and Centricon ®-100 filtration took place. Our experience indicated that the AmpFlSTR ® MiniFiler™ used as a complement to the AmpFlSTR ® Identifiler Plus™ Kit forms an extremely sensitive multiplex STR amplification system. It has been successfully used to obtain multilocus STR profiles from bone samples with minimal amounts of human DNA. We report on the genotyping results obtained from each kit and the biomathematical results of paternity testing.

Standardization of DNA isolation from low cell numbers for chimerism analysis by PCR of short tandem repeats

Analysis of short tandem repeats (STR) by PCR analysis is routinely used in chimerism diagnostics to monitor donor engraftment and to diagnose relapse. Some applications require chimerism analysis of low cell numbers, but no standardized protocol is available for DNA isolation from 1000 to 30,000 cells. The EU-supported EuroChimerism Consortium (project QLRT-2001-01485) selected four different protocols for 'small-scale' DNA isolation, which were tested by six laboratories for their ability to recover reproducible amounts of good quality DNA, suited for PCR-based STR analysis. The protocols included two direct lysis methods with and without detergents and proteinase K, and two commercial column-based kits. The direct lysis method using detergents and proteinase K showed the highest DNA recovery and the best performance in the multiplex PowerPlex16 STR assay. DNA isolated with this method also showed the highest sensitivity in chimerism analysis using singleplex PCR reactions of EuroChimerism STR markers. Sensitivity was reached ranging from 1 to 20% of recipient cells in a donor background. In conclusion, the direct lysis method using detergents and proteinase K is a standardized DNA isolation method well suited for chimerism studies on low cell numbers.

Identification of tumor specimens by DNA analysis in a case of histocytological paraffin tissue block swapping

We report on a patient who was diagnosed with high-grade breast carcinoma by all the pre-surgery clinical evidence of malignancy, but histopathological reports did not reveal any such tumor residue in the post-surgical tissue block. This raised a suspicion that either exchange of block, labeling error, or a technical error took place during gross examination of the tissue. The mastectomy residue was unprocurable to sort out the problem. So, two doubtful paraffin blocks were sent for DNA fingerprinting analysis. The partial DNA profiles (8-9/15 loci) were obtained from histocytological blocks. The random matching probability for both the paraffin blocks and the patient’s blood were found to be 1 in 4.43E4, 1.89E6, and 8.83E13, respectively for Asian population. Multiplex short tandem repeat analysis applied in this case determined that the cause of tumor absence was an error in gross examination of the post-surgical tissue. Moreover, the analysis helped in justifying the therapy given to the patient. Thus, with DNA fingerprinting technique, it was concluded that there was no exchange of the blocks between the two patients operated on the same day and the treatment given to the concerned patient was in the right direction.

Multiplex Short Tandem Repeat Amplification of Low Template DNA Samples with the Addition of Proofreading Enzymes*

With <100 pg of template DNA, routine short tandem repeat (STR) analysis often fails, resulting in no or partial profiles and increased stochastic effects. To overcome this, some have investigated preamplification methods that include the addition of proofreading enzymes to the PCR cocktail. This project sought to determine whether adding proofreading polymerases directly in the STR amplification mixture would improve the reaction when little template DNA is available. Platinum Taq High Fidelity and GeneAmp High Fidelity were tested in Profiler Plus™ STR reactions alone and in combination with AmpliTaq(®) Gold. All reactions included the additional step of a post-PCR purification step. With both pristine low template DNA and casework samples, the addition of these polymerases resulted in comparable or no improvement in the STR amplification signal. Further, stochastic effects and artifacts were observed equally across all enzyme conditions. Based on these studies, the addition of these proofreading enzymes to a multiplex STR amplification is not recommended for low template DNA work.

Development and Validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: A Multiplex Assay for the Direct Amplification of Single-Source Samples*,†

The AmpFℓSTR(®) Identifiler(®) Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single-source blood and buccal samples on FTA(®) card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay's sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA(®) cards, and the assay's specificity was verified by establishing minimal cross-reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA(®) substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler(®) Direct Kit for forensic standards and database samples genotyping.

Assessing a novel room temperature DNA storage medium for forensic biological samples

The ability to properly collect, analyze and preserve biological stains is important to preserving the integrity of forensic evidence. Stabilization of intact biological evidence in cells and the DNA extracts from them is particularly important since testing is generally not performed immediately following collection. Furthermore, retesting of stored DNA samples may be needed in casework for replicate testing, confirmation of results, and to accommodate future testing with new technologies. A novel room temperature DNA storage medium, SampleMatrix™ (SM; Biomatrica, Inc., San Diego, CA), was evaluated for stabilizing and protecting samples. Human genomic DNA samples at varying amounts (0.0625–200 ng) were stored dry in SM for 1 day to 1 year under varying conditions that included a typical ambient laboratory environment and also through successive freeze–thaw cycles (3 cycles). In addition, spiking of 1–4× SM into samples prior to analysis was performed to determine any inhibitory effects of SM. Quantification of recovered DNA following storage was determined by quantitative PCR or by agarose gel electrophoresis, and evaluation of quantitative peak height results from multiplex short tandem repeat (STR) analyses were performed to assess the efficacy of SM for preserving DNA. Results indicate no substantial differences between the quality of samples stored frozen in liquid and those samples maintained dry at ambient temperatures protected in SM. For long-term storage and the storage of low concentration samples, SM provided a significant advantage over freezer storage through higher DNA recovery. No detectable inhibition of amplification was observed at the recommended SM concentration and complete profiles were obtained from genomic DNA samples even in the presence of higher than recommended concentrations of the SM storage medium. The ability to stabilize and protect DNA from degradation at ambient temperatures for extended time periods could have tremendous impact in simplifying and improving sample storage conditions and requirements. The current work focuses on forensics analysis; however this technology is applicable to all endeavors requiring storage of DNA.

Development and characterization of two mini-X chromosomal short tandem repeat multiplexes

This study presents the development and characterization of two X chromosomal short tandem repeat (STR) multiplexes utilizing reduced-size amplicons (less than 200 base pairs) for identity and kinship testing with degraded DNA. Approximately 1360 samples across 4 U.S. population groups were typed for 15 X chromosomal STR markers: DXS6789, DXS7130, DXS9902, GATA31E08, DXS7424, GATA165B12, DXS101, DXS6795, GATA172D05, DXS10147, DXS8378, DXS7132, DXS6803, HPRTB, and DXS7423. A high degree of polymorphism was observed for each marker and both multiplexes were sensitive down to 200 pg of pristine DNA. The two proposed multiplexes are suitable for forensic use, and show potential for improved analysis of compromised bone samples.

GeneMarker® HID: A Reliable Software Tool for the Analysis of Forensic STR Data

GeneMarker(®) HID was assessed as a software tool for the analysis of forensic short tandem repeat (STR) data and as a resource for analysis of custom STR multiplexes. The software is easy to learn and use, and includes design features that have the potential to reduce user fatigue. To illustrate reliability and accuracy, STR data from both single-source and mixture profiles were analyzed and compared to profiles interpreted with another software package. A total of 1898 STR profiles representing 28,470 loci and more than 42,000 alleles were analyzed with 100% concordance. GeneMarker HID was also used to successfully analyze data generated from a custom STR multiplex, with simplified and rapid implementation. Finally, the impact of the user-friendly design features of the software was assessed through a time scale study. The results suggest that laboratories can reduce the time required for data analysis by at least 25% when using GeneMarker HID.

Population genetic analyses of the NGM STR loci

The AmpFlSTR® NGM™ PCR Amplification Kit enables amplification of 15 autosomal short tandem repeat (STR) loci. The loci are the ten STRs in the SGM Plus® Kit plus the EDNAP and ENSFI recommended STRs D10S1248, D22S1045, D2S441, D1S1656, and D12S391. Allele frequency and other forensically relevant statistics data were generated for the NGM loci in three US population groups (African Americans, Caucasians, and Hispanics). The analyses support that the NGM multiplex is one of the most informative STR multiplex kits available to the forensic science community. At the population level, there are no more detectable departures from expectations of the independence of alleles within as well as between loci than would be expected due to chance, even for the two syntenic loci vWA and D12S391; however, linkage analysis in three large pedigree families shows close linkage between these two loci with a recombination fraction of 0.108. Therefore, in contrast to the practices in calculating the rarity of a DNA profile, for kinship analyses independence between the loci, vWA and D12S391 cannot be assumed.

Integrated Microfluidic System for Rapid Forensic DNA Analysis: Sample Collection to DNA Profile

We demonstrate a conduit for the delivery of a step change in the DNA analysis process: A fully integrated instrument for the analysis of multiplex short tandem repeat DNA profiles from reference buccal samples is described and is suitable for the processing of such samples within a forensic environment such as a police custody suite or booking office. The instrument is loaded with a DNA processing cartridge which incorporates on-board pumps and valves which direct the delivery of sample and reagents to the various reaction chambers to allow DNA purification, amplification of the DNA by PCR, and collection of the amplified product for delivery to an integral CE chip. The fluorescently labeled product is separated using micro capillary electrophoresis with a resolution of 1.2 base pairs (bp) allowing laser induced fluorescence-based detection of the amplified short tandem repeat fragments and subsequent analysis of data to produce a DNA profile which is compatible with the data format of the UK DNA database. The entire process from taking the sample from a suspect, to database compatible DNA profile production can currently be achieved in less than 4 h. By integrating such an instrument and microfluidic cartridge with the forensic process, we believe it will be possible in the near future to process a DNA sample taken from an individual in police custody and compare the profile with the DNA profiles held on a DNA Database in as little as 3 h.

WITHDRAWN: Development and Characterization of Two Mini-X Chromosomal Short Tandem Repeat Multiplexes

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Rapid amplification of commercial STR typing kits

Forensic DNA typing is currently conducted in approximately 8–10 h. The process includes DNA extraction, quantitation, multiplex PCR amplification, and fragment length detection. Today's commercial multiplex short tandem repeat (STR) typing kits are not optimized for rapid PCR thermal cycling. Current protocols require approximately 3 h for amplifying a multiplex containing 15 STR loci plus amelogenin. With the continuing development of miniaturization technologies such as microfluidic and micro-capillary devices, there is a desire to reduce the overall time required to type DNA samples. Such miniature devices could be used for initial screening at a crime scene, at a border, and at airports. There is also the benefit of reducing the required PCR amplification time for labs typing single-source reference samples. Surveys of fast processing polymerases working in combination with rapid cycling protocols have resulted in the development of a ‘rapid’ PCR amplification protocol. Results are obtained in less than 36 min run on a standard peltier-based thermal cycler employing a heating rate of 4 °C/s. Capillary electrophoresis characterization of the PCR products indicates good peak balance between loci, strong signal intensity and minor adenylation artifacts. Genotyping results are concordant with standard amplification conditions utilizing a standard 3 h (non-rapid) thermal cycling procedure. The rapid assay conditions are robust enough to routinely amplify 0.5 ng of template DNA (with 28 cycles).

A 26plex Autosomal STR Assay to Aid Human Identity Testing*†

A short tandem repeat multiplex assay has been successfully developed with 25 autosomal loci plus the sex-typing locus amelogenin for a total of 26 amplified products in a single reaction. Primers for the loci were designed so that all of the amplicons present were distributed from 65 base pairs (bp) to less than 400 bp within a five-dye chemistry design with the fifth dye reserved for the sizing standard. A multiplex design strategy was developed to overcome challenges encountered in creating this assay. The limits of the multiplex were tested, resulting in the successful amplification of a wide range of genomic DNA sample concentrations from 2 ng to as low as 100 pg with 30 cycles of PCR. The 26plex has the potential to benefit the forensic community for reference sample testing and complex relationship evaluation.

Factor V Leiden in an Urartian, Dating Back to 1000 BC

Factor V Leiden (FVL) is the most common monogenic disorder that causes activated protein C (APC) resistance, creating hyper-coagulation. The mutation shows an uneven geographic distribution, significantly high in European populations. The mutation is believed to have originated approximately 20 000 years ago probably from a geographic region close to Anatolia. This fact makes it noteworthy to search for the mutation in ancient populations that once lived in this area. One of these civilizations, Urartu was centered around Van Lake in Eastern Turkey. The archeological remains from the excavations of the region are dated back to 1000 BC. Teeth, taken from the excavations of Van Yoncatepe fortress, were taken into DNA analysis considering all the precautions for ancient DNA analysis. Multiplex STR (Short Tandem Repeats) analysis were performed both to determine the gender of the samples and to conclude that the samples are preserved from modern DNA contamination. After getting an 80% amplification success for amelogenin, a melting curve analysis using lightcycler was performed to determine the FVL genotype of each sample. Of the 60 samples, 1 gave a positive amplification result for FV gene and was found to be heterozygous. To date, the age of this mutation was estimated based on statistical calculations using haplotype frequencies; here for the first time, we report FVL in an ancient population of 3000 years.

Canine Population Data Generated from a Multiplex STR Kit for Use in Forensic Casework*

Canine biological specimens are often part of the physical evidence from crime scenes. Until now, there have been no validated canine-specific forensic reagent kits available. A multiplex genotyping system, comprising 18 short tandem repeats (STRs) and a sex-linked zinc finger locus for gender determination, was developed for generating population genetic data assessing the weight of canine forensic DNA profiles. Allele frequencies were estimated for 236 pedigreed and 431 mixed breed dogs residing in the U.S. Average random match probability is 1 in 2 x 10(33) using the regional database and 1 in 4 x 10(39) using the breed dataset. Each pedigreed population was genetically distinct and could be differentiated from the mixed breed dog population but genetic variation was not significantly correlated with geographic transition. Results herein support the use of the allele frequency data with the canine STR multiplex for conveying the significance of identity testing for forensic casework, parentage testing, and breed assignments.