Multiplex short tandem repeat amplification of low template DNA samples with the addition of proofreading enzymes

Abstract:  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.     

Simulated approach to estimate the number and combination of known/unknown contributors in mixed DNA samples using 15 short tandem repeat loci

The calculation of likelihood ratios (LRs) for DNA mixture analysis is necessary to establish an appropriate hypothesis based on the estimated number of contributors and known contributor genotypes. In this paper, we recommend a relevant analytical method from the 15 short tandem repeat typing system (the Identifiler multiplex), which is used as a standard in Japanese forensic practice and incorporates a flowchart that facilitates hypothesis formulation. We postulate that: (1) all detected alleles need to be above the analytical threshold (e.g., 150 relative fluorescence unit (RFU)); (2) alleles of all known contributors should be detected in the mixture profile; (3) there should be no contribution from close relatives. Furthermore, we deduce that mixtures of four or more persons should not be interpreted by Identifiler as the LR values of 100,000 simulated cases have a lower expectation of exceeding our temporal LR threshold (10,000) which strongly supports the prosecution hypothesis. We validated the method using various computer-based simulations. The estimated number of contributors is most likely equal to the actual number if all alleles detected in the mixture can be assigned to those from the known contributors. By contrast, if an unknown contributor(s) needs to be designated, LRs should be calculated from both two-person and three-person contributions. We also consider some cases in which the unknown contributor(s) is genetically related to the known contributor(s).     

Characterization and validation studies of powerPlex 2.1, a nine-locus short tandem repeat (STR) multiplex system and penta D monoplex

In order to increase the power of discrimination for human identification purposes, a nine-locus short tandem repeat (STR) multiplex, the GenePrint PowerPlex 2.1 system (PowerPlex 2.1) developed by Promega Corporation and a separate pentanucleotide-repeat locus, Penta D, were tested. This megaplex system includes the highly polymorphic loci FGA, TPOX, D8S1179, vWA, Penta E, D18S51, D21S11, TH01, and D3S1358 and may be used in combination with the eight-locus STR multiplex, the GenePrint PowerPlex 1.1 system (PowerPlex 1.1) that has been previously developed. Three of the loci, TPOX, TH01 and vWA, have been included in both systems for quality control purposes. As with PowerPlex 1.1, PowerPlex 2.1 is also based on a two-color detection of fluorescent-labeled DNA products amplified by polymerase chain reaction (PCR) and provides a valuable tool for accurate and rapid allele determination. The primer sequences used in the PowerPlex 2.1/Penta D system are also presented in this report. To meet the "Quality Assurance Standards for Forensic DNA Testing Laboratories" (FBI), we tested the efficiency and reproducibility of the PowerPlex 2.1/PentaD system by several validation studies that were conducted as a joint project among seven laboratories. Validation tests included concordance studies, sensitivity, and species specificity determination, as well as performance in forensic and environmentally impacted samples. The results produced from these tests demonstrated the consistency and reliability of the PowerPlex 2.1/Penta D system.     

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 of a pentaplex X-chromosomal short tandem repeat typing system and population genetic studies

Quadruplex and pentaplex systems for polymerase chain reaction amplification of X-chromosomal short tandem repeats DXS101, HPRTB, DXS8377, DXS981 (STRX1) and DXS6789 were developed for automated profiling of liquid and membrane-bound DNA samples. Chinese, Japanese and Thai populations were typed using a quadruplex system, while German and Philippine populations were analyzed using a five-locus system. Out of 88 meioses studied in Philippine family samples at each locus, a possible one repeat deletion (allele 51 to 50) at DXS8377 was observed in a father-daughter pair. Exact tests performed on genotype data from females in the Philippine, German and Thai populations indicated that these groups conform to Hardy-Weinberg equilibrium. Exact tests for population differentiation indicate significant variations in allele distributions, particularly at loci DXS101, DXS981 and DXS6789. Considered individually, DXS8377 was the most polymorphic and HPRTB the least polymorphic locus in these five populations. When the forensic efficiency of the quadruplex system was calculated, the combined power of discrimination among males (PD(M)) was no lower than 0.998, while among females the combined PD(F) was at least 0.9999 in all populations. The combined power of paternity exclusion was a minimum of 0.998 in trio cases and 0.98 in motherless cases. The addition of locus DXS6789 to the German and Philippine population databases using a pentaplex increased the forensic efficiency of the analysis system.     

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.     

Autosomal short tandem repeat analysis of ancient DNA by coupled use of mini- and conventional STR kits

Abstract: Multiplex autosomal short tandem repeat (STR) genotyping enables researchers to obtain genetic information from ancient human samples. In this study, we tested newly developed AmpF?STR^® MiniFiler? kit for autosomal STR analysis of ancient DNA (aDNA), using human femurs (n = 8) collected from medieval Korean tombs. After extracting aDNA from the bones, autosomal STR analyses were repeated for each sample using the AmpF?STR^® MiniFiler? and Identifiler? kits. Whereas only 21.87% of larger‐sized loci profiles could be obtained with the Identifiler? kit, 75% of the same loci profiles were determined by MiniFiler? kit analysis. This very successful amplification of large‐sized STR markers from highly degraded aDNA suggests that the MiniFiler? kit could be a useful complement to conventional STR kit analysis of ancient samples.     

Usefulness of conventional blood groups, DNA-minisatellites, and short tandem repeat polymorphisms in paternity testing: a comparison.

A total of 215 paternity cases were analysed after testing 24 marker systems. Despite technical advantages of polymerase chain reaction related polymorphisms (automatisation, employment of robots, lesser requirements concerning of quality and quantity of DNA) it could be shown that the exclusive employment of a parentage testing kit is compromised by an increased risk of erroneous conclusions. It is estimated that in about 3-4% of the cases ambiguous situations have to be expected which are caused by the occurrence of single or double exclusions. In these cases it is impossible to decide whether the exclusions indicate either true nonpaternity or a de novo mutation. The situation might become even more complicated if an involvement of a close relative of the alleged father cannot be ruled out. We cautiously advance the hypothesis that in parentage testing DNA minisatellite polymorphisms from an optimal set of tools.     

Italian population data on two new short tandem repeat loci: D2S1338 and Penta E

A population study on two new short tandem repeat (STR) loci D2S1338 (a tetranucleotide repeat) and Penta E (a pentanucleotide repeat) was performed on 208 unrelated Italian Caucasians. The DNA was amplified by polymerase chain reaction (PCR) and separation and detection of the amplified STR fragments were carried out by use of a PE/ABD PRISM 377 DNA Sequencer 377 automated system (Applied Biosystems Division/Perkin-Elmer). Both loci meet Hardy-Weinberg expectations. There is no evidence for departures from expectations between the two loci. The combined Probability of Discrimination and Probability of Exclusion for the two STR loci are 0.999155 and 0.944925, respectively. The results demonstrate that these two regions can be useful for differentiating among individuals, particularly in concert with other STR loci.     

Chelating resin-based extraction of DNA from dental pulp and sex determination from incinerated teeth with Y-chromosomal alphoid repeat and short tandem repeats

A procedure utilizing Chelex 100, chelating resin, was adapted to extract DNA from dental pulp. The procedure was simple and rapid, involved no organic solvents, and did not require multiple tube transfers. The extraction of DNA from dental pulp using this method was as efficient, or more so, than using proteinase K and phenol-chloroform extraction. In this study, the Chelex method was used with amplification and typing at Y-chromosomal loci to determine the effects of temperature on the sex determination of the teeth. The extracted teeth were incinerated in a dental furnace for 2 minutes at 100 degrees C, 200 degrees C, 300 degrees C, 400 degrees C, and 500 degrees C. After the isolation of DNA from the dental pulp by the Chelex method, alphoid repeats, and short tandem repeats, the human Y chromosome (DYZ3), DYS19, SYS389, DYS390, and DYS393 could be amplified and typed in all samples incinerated at up to 300 degrees C for 2 minutes. The DYS389 locus in some samples could not be amplified at 300 degrees C for 2 minutes. An autopsy case is described in which genotypings of DYS19, DYS390, and DYS393 from dental pulp obtained from a burned body were needed. The data presented in this report suggest that Chelex 100-based DNA extraction, amplification, and typing are possible in burned teeth in forensic autopsy cases.     

Combining autosomal and Y-chromosomal short tandem repeat data in paternity testing with male child: methods and application

Paternity testing is being increasingly requested with the aim of challenging presumptive fatherhood. The ability to establish the biological father is usually based on the genotyping of autosomal short tandem repeat (STR) in alleged father, mother and child, but the use of Y-chromosomal STR has gained interest in the last few years. In this work, we propose a new probabilistic approach that combines autosomal and Y-chromosomal STR data in paternity testing with father/son pairs taking into account mutation events. We also suggest a new two-stage approach where we first type Y-STRs and possibly autosomal STR for the putative father and son, conditional on Y-STR results. We applied this approach to 22 cases. Our results show that Y-STRs can identify nonpaternity cases with high accuracy but need to be validated with autosomal STR to establish paternity. Moreover, the two-stage approach is less costly than the standard approach and is very useful in motherless cases.     

Identification of exhumed remains of fire tragedy victims using conventional methods and autosomal/Y-chromosomal short tandem repeat DNA profiling

In a fire tragedy in Manila in December 1998, one of the worst tragic incidents which resulted in the reported death of 23 children, identity could not be established initially resulting in the burial of still unidentified bodies. Underscoring the importance of identifying each of the human remains, the bodies were exhumed 3 months after the tragedy. We describe here our work, which was the first national case handled by local laboratories wherein conventional and molecular-based techniques were successfully applied in forensic identification. The study reports analysis of DNA obtained from skeletal remains exposed to conditions of burning, burial, and exhumation. DNA typing methods using autosomal and Y-chromosomal short tandem repeat (Y-STR) markers reinforced postmortem examinations using conventional identification techniques. The strategy resulted in the identification of 18 out of the 21 human remains analyzed, overcoming challenges encountered due to the absence of established procedures for the recovery of mass disaster remains. There was incomplete antemortem information to match the postmortem data obtained from the remains of 3 female child victims. Two victims were readily identified due to the availability of antemortem tissues. In the absence of this biologic material, parentage testing was performed using reference blood samples collected from parents and relatives. Data on patrilineal lineage based on common Y-STR haplotypes augmented autosomal DNA typing, particularly in deficiency cases.     

自动荧光分析法检验陈旧骨骼和牙齿的vWA和LPL位点

为鉴定陈旧骨骼和牙齿的尸源 ,对DNA的提取方法进行了研究,并建立了vWA和LPL位点的自动荧光分析技术,该法简便、快速、有效、可对存放2、3、4、7年的陈旧骨骼和牙齿成功地进行检验,在检验中能直接确定样品的等位基因     

Paternity testing: blood group systems and DNA analysis by variable number of tandem repeat markers

Two recent paternity cases are reported. In the first case of paternity exclusion, deoxyribonucleic acid (DNA) restriction fragment length polymorphisms (RFLPs) on variable number of tandem repeat (VNTR) loci with multiple alleles were informative, as well as established systems of red blood antigens, red cell enzymes, serum proteins, and human leukocyte antigens. In the second case, in which both the alleged father and the first wife were deceased, the paternal genotype was determined by using genetic markers from the second wife and four children, which then were compared with the paternal alleles of the child in question, the plaintiff in this case. The high probability of paternity (0.999,998,7) made us conclude that the man probably was the actual father. The DNA analysis by VNTR probes appears to be quite valuable in the study of paternity cases.     

Allelic distribution of nine short tandem repeat (STR), HLA-DQA1, and polymarker loci in an Omani sample population

Allele frequency distributions of nine short tandem repeat (STR) loci, D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D7S820, and D13S317, HLA-DQA1 and polymarker (PM) loci were studied in a sample population from Sultanate of Oman, Middle East. Blood samples were collected from 162 unrelated individuals. For all STR, HLA-DQA1 and PM loci, no deviations from Hardy-Weinberg equilibrium, based on the exact test, were observed. The most discriminating loci were D18S51 (PD=0.966) and FGA (PD=0.964), while the least informative locus is GYPA (PD=0.585). The allele frequency data may be useful in forensic case work.     

Fingerprint enhancement revisited and the effects of blood enhancement chemicals on subsequent profiler Plus fluorescent short tandem repeat DNA analysis of fresh and aged bloody fingerprints

This study was aimed at determining the effect of seven blood enhancement reagents on the subsequent Profiler Plus fluorescent STR DNA analysis of fresh or aged bloody fingerprints deposited on various porous and nonporous surfaces. Amido Black, Crowle's Double Stain. 1,8-diazafluoren-9-one (DFO), Hungarian Red, leucomalachite green, luminol and ninhydrin were tested on linoleum, glass, metal, wood (pine, painted white), clothing (85% polyester/15% cotton, 65% polyester/35% cotton, and blue denim) and paper (Scott 2-ply and Xerox-grade). Preliminary experiments were designed to determine the optimal blood dilutions to use to ensure a DNA typing result following chemical enhancement. A 1:200 blood dilution deposited on linoleum and enhanced with Crowle's Double Stain generated enough DNA for one to two rounds of Profiler Plus PCR amplification. A comparative study of the DNA yields before and after treatment indicated that the quantity of DNA recovered from bloody fingerprints following enhancement was reduced by a factor of 2 to 12. Such a reduction in the DNA yields could potentially compromise DNA typing analysis in the case of small stains. The blood enhancement chemicals selected were also evaluated for their capability to reveal bloodmarks on the various porous and nonporous surfaces chosen in this study. Luminol. Amido Black and Crowle's Double Stain showed the highest sensitivity of all seven chemicals tested and revealed highly diluted (1:200) bloody fingerprints. Both luminol and Amido Black produced excellent results on both porous and nonporous surfaces, but Crowle's Double Stain failed to produce any results on porous substrates. Hungarian Red, DFO, leucomalachite green and ninhydrin showed lower sensitivities. Enhancement of bloodmarks using any of the chemicals selected, and short-term exposure to these same chemicals (i.e., less than 54 days), had no adverse effects on the PCR amplification of the nine STR systems surveyed (D3S 1358, HumvWA, HumFGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820) or of the gender determination marker Amelogenin. The intensity of the fluorescent signals was very similar and the allele size measurements remained constant and identical to those of untreated bloody fingerprints. No additional background fluorescence was noted. Continuous exposure (for 54 days) to two of the seven enhancement chemicals selected (i.e., Crowle's Double Stain and Hungarian Red) slightly reduced the amplification efficiency of the longer STR loci in profiles of fresh and 7 to 14-day-old bloodprints. This suggests that long-term exposure to these chemicals possibly affects the integrity of the DNA molecules. This study indicates that significant evidence can be obtained from fresh or aged bloody fingerprints applied to a variety of absorbent and nonabsorbent surfaces which are exposed to different enhancement chemicals for short or long periods of time. It also reaffirms that PCR STR DNA typing procedures are robust and provide excellent results when used in concert with fluorescence-based detection assays after fingerprint identification has taken place.     

A duplex real-time qPCR assay for the quantification of human nuclear and mitochondrial DNA in forensic samples: implications for quantifying DNA in degraded samples

A duplex real-time qPCR assay was developed for quantifying human nuclear and mitochondrial DNA in forensic samples. The nuclear portion of the assay utilized amplification of a approximately 170-190 bp target sequence that spans the repeat region of the TH01 STR locus, and the mitochondrial portion of the assay utilized amplification of a 69 bp target sequence in the ND1 region. Validation studies, performed on an ABI 7000 SDS instrument using TaqMan detection, demonstrated that both portions of the duplex assay provide suitable quantification sensitivity and precision down to 10-15 copies of each genome of interest and that neither portion shows cross-reactivity to commonly encountered non-human genomes. As part of the validation studies, a series of DNase-degraded samples were quantified using three different methods: the duplex nuclear-mitochondrial qPCR assay, the ABI Quantifiler Human DNA Quantification Kit qPCR assay, which amplifies and detects a 62 bp nuclear target sequence, and slot blot hybridization. For non-degraded and moderately degraded samples in the series, all three methods were suitably accurate for quantifying nuclear DNA to achieve successful STR amplifications to yield complete profiles using the ABI AmpFlSTR Identifiler kit. However, for highly degraded samples, the duplex qPCR assay provided better estimates of nuclear template for STR amplification than did either the commercial qPCR assay, which overestimated the quantity of STR-sized DNA fragments, leading to an increased proportion of undetected alleles at the larger STR loci, or slot blot hybridization, which underestimated the quantity of nuclear DNA, leading to an increased proportion of STR amplification artifacts due to amplification of excess template.     

Population data at two short tandem repeat loci D2S1338 and D19S433 in the sample of multinational Bosnia and Herzegovina residents

POPULATION: We have analyzed the distribution of allele frequencies at two short tandem repeats loci (D2S1338 and D19S433) in a multinational sample of Bosnia and Herzegovina (B&H) residents. A total of 110 unrelated male and female individuals (Caucasians) from different regions of B&H were sampled for the analysis. We ensured that the sample reflected approximate proportional participation of the three main ethnic groups in the population of B&H (Bosniacs-Muslim [45%], Serbs [34%], Croats [21%]).     

Developmental validation of the AmpF?STR SEfiler Plus™ PCR amplification kit: An improved multiplex with enhanced performance for inhibited samples

Since its introduction in 2002, the AmpF?STR^® SEfiler™ kit has provided a highly discriminating DNA profiling option to German forensic laboratories by combining the widely used SGM Plus^® Kit loci with the SE-33 locus required for the German DNA Database. Whilst proving successful on database samples, laboratories using the SEfiler™ kit have reported the need for chemistry better able to handle the ever-increasing number of casework samples.The new AmpF?STR^® SEfiler Plus™ kit contains the same loci and primer sequences as the SEfiler™ kit but uses improved synthesis and purification processes to minimize the presence of dye-labeled artifacts. Other improvements include modified PCR cycling conditions for enhanced sensitivity and a new buffer formulation that improves performance with inhibited samples when compared to the original SEfiler™ kit.Validation studies demonstrating the effectiveness of the multiplex are presented with emphasis on the models of inhibition and casework samples.