STR analysis of degraded DNA using a miniplex

Analysis of short tandem repeat (STR) markers currently represents the most useful instrument in the field of forensic genetics. The problem with forensic material is the degradation of the sample material. In recent years, several papers have demonstrated that short amplicon STR (miniSTR) represents one of the most useful tools for analyzing degraded DNA samples.In the present study, we attempted to develop a short amplicon STR multiplex system (autosomal and y-chromosomal) for analyzing degraded DNA using some newly designed primer sets for a multiplex polymerase chain reaction (PCR) systems for typing.An assay of degraded DNA samples using the designed multiplex systems, including artificially degraded samples and degraded forensic casework samples, proved remarkably effective. Comparing the multiplex with commercial kits, first results show a well success rate.     

Validation and casework testing of the BioPlex-11 for STR typing of telogen hair roots

A new STR typing strategy has been developed allowing the simultaneous amplification and subsequent analysis of 11 polymorphic systems with amplicon sizes smaller than 270bp. The multiplex amplification reaction includes six STR loci from the European standard set of loci (ESS) for DNA databases (D3S1358, D8S1179, D21S11, THO1, FGA and VWA) as well as four additional STR systems selected for their robustness (D2S1338, D12S391, TPOX and D5S818) together with the sex-specific locus amelogenin. After PCR amplification, the multiplex reaction is splitted into two sets of STR multiplexes by using biotin labelled primers only for one set. Using streptavidin-coated Sepharose beads five STR systems are separated from the other six systems prior to being analysed in two different runs on a capillary gel electrophoresis instrument. The multiplex system was developed and tested especially for the use in forensic casework if only limited amounts or highly degraded DNA is available, for instance, when isolated from telogen hair roots.     

The effectiveness of various strategies to improve DNA analysis of formaldehyde-damaged tissues from embalmed cadavers for human identification purposes

Formalin-fixed tissues provide the medical and forensic communities with alternative and often last resort sources of DNA for identification or diagnostic purposes. The DNA in these samples can be highly degraded and chemically damaged, making downstream genotyping using short tandem repeats (STRs) challenging. Therefore, the use of alternative genetic markers, methods that pre-amplify the low amount of good quality DNA present, or methods that repair the damaged DNA template may provide more probative genetic information. This study investigated whether whole genome amplification (WGA) and DNA repair could improve STR typing of formaldehyde-damaged (FD) tissues from embalmed cadavers. Additionally, comparative genotyping success using bi-allelic markers, including INDELs and SNPs, was explored. Calculated random match probabilities (RMPs) using traditional STRs, INDEL markers, and two next generation sequencing (NGS) panels were compared across all samples. Overall, results showed that neither WGA nor DNA repair substantially improved STR success rates from formalin-fixed tissue samples. However, when DNA from FD samples was genotyped using INDEL and SNP-based panels, the RMP of each sample was markedly lower than the RMPs calculated from partial STR profiles. Therefore, the results of this study suggest that rather than attempting to improve the quantity and quality of severely damaged and degraded DNA prior to STR typing, a more productive approach may be to target smaller amplicons to provide more discriminatory DNA identifications. Furthermore, an NGS panel with less loci may yield better results when examining FD samples, due to more optimized chemistries that result in greater allelic balance and amplicon coverage.     

Analysis of mitochondrial SNPs in addition to conventional STR-typing in a case of aggravated theft

In the field of forensic DNA typing, the analysis of Short Tandem Repeats (STRs) can fail in cases of degraded DNA. The typing of coding region Single Nucleotide Polymorphisms (SNPs) of the mitochondrial genome provides an approach to acquire additional information. In the examined case of aggravated theft, both suspects could be excluded of having left the analyzed hair on the crime scene by SNP typing. This conclusion was not possible subsequent to STR typing. SNP typing of the trace on the torch light left on the crime scene increased the likelihood for suspect no. 2 to be the origin of this trace. This finding was already indicated by STR analysis. Suspect no. 1 was excluded for being the origin of this trace by SNP typing which was also indicated by STR analysis. A limiting factor for the analysis of SNPs is the maternal inheritance of mitochondrial DNA. Individualisation is not possible. In conclusion, it can be said that in the case of traces which cause problems with conventional STR typing the supplementary analysis of coding region SNPs from the mitochondrial genome is very reasonable and greatly contributes to the refinement of analysis methods in the field of forensic genetics.     

The application of miniplex primer sets in the analysis of degraded DNA from human skeletal remains

A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280 base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Powerplex 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200 bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.     

Significance of mitochondrial DNA for forensic stain analysis, identification and forensic lineage determination

DNA testing using conventional STR systems may produce insufficient results, if the genomic DNA in the specimen is either highly degraded or the available quantity is very small (e.g. skin particles, hair shafts or ancient bones). In some of these cases the examination of mitochondrial DNA, which is present in considerably larger copy numbers in the cytoplasm, is more successful than that of nuclear DNA. Identification of unknown corpses by conventional DNA typing sometimes remains doubtful, if only samples from presumably distant relatives or putative brothers or sisters are available for comparison. Since mitochondrial DNA is generally transmitted in maternal lineages, its sequence pattern can be directly compared with those of other individuals and, in case of the same maternal lineage, corresponding sequence chromatograms are to be expected. In connection with nuclear DNA typing methods certain sequence motives may furnish clues to ethnic groups. The report presents three cases illustrating the application possibilities of mtDNA typing in forensic practice.     

The Successful Recovery of Low Copy Number and Degraded DNA from Bones Exposed to Seawater Suitable for Generating a DNA STR Profile

A universal method allowing for DNA profiling from bones exposed to seawater has not been reported yet. This study refers on the identification of a body immersed in seawater for 8 months. The biological material for identification was the mandibular body, usually characterized by low success rates of DNA analysis. Initially, two extraction protocols were performed with negative results: one used for bones immersed in fresh water and a silica‐column procedure. A third protocol was performed, which combined the extraction of a higher amount of bone powder, the use of multi‐silica‐based extraction columns followed by a concentration step. This protocol allowed to obtain low copy number DNA and to generate a 12‐loci STR profile by combining conventional STR typing and mini‐STR technologies. This protocol could be suitable when human bones have been exposed to severe environmental conditions, and the available nuclear DNA is highly degraded and in low copy number.     

Validation of the Short Amplicon Multiplex Q8 Including the German DNA Database Systems*

Abstract: We have developed a concept to enable the analyzing of degraded stains with limited DNA template quantity. Therefore we have constructed a short tandem repeat (STR) multiplex including the German DNA database systems (Q8). The amplicon lengths are smaller than 280 bp. For the validation of Q8 over 50 degraded samples were investigated. Amplifications were performed with “low copy number” PCR, the number of PCR cycles was increased to 33 and the reaction volume was decreased to 12.5 μL. Compared with the MPX2 and Nonaplex kit, the average success rate was increased using the Q8 kit by approximately 20% and 30%, respectively. The efficiency of a sensitive STR multiplex with reduced amplicon lengths was confirmed in comparing the success rates of Q8 for typing degraded samples and samples with limited amount of DNA template while partial profiles were observed with the majority of the samples using commercially available kits.     

Tri-allelic SNP markers enable analysis of mixed and degraded DNA samples

For the analysis of degraded DNA in disaster victim identification (DVI) and criminal investigations, single nucleotide polymorphisms (SNPs) have been recognized as promising markers mainly because they can be analyzed in short sized amplicons. Most SNPs are bi-allelic and are thereby ineffective to detect mixtures, which may lead to incorrect genotyping. We developed an algorithm to find non-binary (i.e. tri-allelic or tetra-allelic) SNPs in the NCBI dbSNP database. We selected 31 potential tri-allelic SNPs with a minor allele frequency of at least 10%. The tri-allelic nature was confirmed for 15 SNPs residing on 14 different chromosomes. Multiplex SNaPshot™ assays were developed, and the allele frequencies of 16 SNPs were determined among 153 Dutch and 111 Netherlands Antilles reference samples. Using these multiplex SNP assays, the presence of a mixture of two DNA samples in a ratio up to 1:8 could be recognized reliably. Furthermore, we compared the genotyping efficiency of the tri-allelic SNP markers and short tandem repeat (STR) markers by analyzing artificially degraded DNA and DNA from 30 approximately 500-year-old bone and molar samples. In both types of degraded DNA samples, the larger sized STR amplicons failed to amplify whereas the tri-allelic SNP markers still provided valuable information. In conclusion, tri-allelic SNP markers are suited for the analysis of degraded DNA and enable the detection of a second DNA source in a sample.     

荧光标记短片段STR复合扩增系统的法医学应用研究

目的解决严重降解(低于300bp)DNA检验问题,提高降解DNA的检出率。方法重新设计引物,减小扩增产物片段长度,用荧光标记短片段STR复合扩增体系进行DNA检验,扩增结果与用Identifiler试剂盒扩增出的结果进行比较。结果用荧光标记短片段STR复合扩增系统对实际案件中的高度降解DNA检材进行检验,可以获得满意分型。结论该系统可用于严重降解DNA检材的检验工作。     

Development and validation of the AmpFlSTR MiniFiler PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA

DNA typing of degraded DNA samples can be a challenging task when using the current commercially available multiplex short tandem repeat (STR) analysis kits. However, the ability to type degraded DNA specimens improves by redesigning current STR marker amplicons such that smaller sized polymerase chain reaction (PCR) products are generated. In an effort to increase the amount of information derived from these types of DNA samples, the AmpFlSTR MiniFiler PCR Amplification Kit has been developed. The kit contains reagents for the amplification of eight miniSTRs which are the largest sized loci in the AmpFlSTR Identifiler PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA). Five of these STR loci (D16S539, D21S11, D2S1338, D18S51, and FGA) also are some of the largest loci in the AmpFlSTR SGM Plus kit. This informative nine-locus multiplex, which includes the gender-identification locus Amelogenin, has been validated according to the FBI/National Standards and SWGDAM guidelines. Our results demonstrate significant performance improvements in models of DNA degradation, PCR inhibition, and nonprobative samples when compared to the AmpFlSTR Identifiler and SGM Plus kits. These data support that the MiniFiler kit will increase the likelihood of obtaining additional STR information from forensic samples in situations in which standard STR chemistries fail to produce complete profiles.     

STR analysis of artificially degraded DNA-results of a collaborative European exercise

Degradation of human DNA extracted from forensic stains is, in most cases, the result of a natural process due to the exposure of the stain samples to the environment. Experiences with degraded DNA from casework samples show that every sample may exhibit different properties in this respect, and that it is difficult to systematically assess the performance of routinely used typing systems for the analysis of degraded DNA samples. Using a batch of artificially degraded DNA with an average fragment size of approx. 200 bp a collaborative exercise was carried out among 38 forensic laboratories from 17 European countries. The results were assessed according to correct allele detection, peak height and balance as well as the occurrence of artefacts. A number of common problems were identified based on these results such as strong peak imbalance in heterozygous genotypes for the larger short tandem repeat (STR) fragments after increased PCR cycle numbers, artefact signals and allelic drop-out. Based on the observations, strategies are discussed to overcome these problems. The strategies include careful balancing of the amount of template DNA and the PCR cycle numbers, the reaction volume and the amount of Taq polymerase. Furthermore, a careful evaluation of the results of the fragment analysis and of automated allele calling is necessary to identify the correct alleles and avoid artefacts.     

Characterization of new miniSTR loci to aid analysis of degraded DNA

A number of studies have demonstrated that successful analysis of degraded DNA specimens from mass disasters or forensic evidence improves with smaller sized polymerase chain reaction (PCR) products. We have scanned the literature for new STR loci, unlinked from the CODIS markers, which can generate amplicons less than 125 bp in size and would therefore be helpful in testing degraded DNA samples. New PCR primers were designed and tested for the STR loci D1S1677, D2S441, D4S2364, D10S1248, D14S1434, and D22S1045, arranged into two miniSTR triplexes. All loci show a moderate degree of polymorphism among 474 U.S. population samples tested and were reliable and sensitive to at least 100 pg of DNA template under controlled laboratory conditions and pristine DNA samples. The utility of these new loci were confirmed in comparing the success of the miniSTR assays for typing degraded bone samples while partial profiles were observed with the majority of the samples using a commercial STR kit.     

Additional sequence characterization of NIST SRM 2391c: PCR-Based DNA Profiling Standard

The NIST Standard Reference Material (SRM) 2391c: PCR-Based DNA Profiling Standard was designed for use in the standardization of forensic and paternity quality assurance procedures for fragment-based typing short tandem repeat (STR) alleles generated by the polymerase chain reaction (PCR). Certified genotypes of the 6 components A–F were assigned for 24 autosomal and 17 Y-STR markers plus Amelogenin using concordance testing between commercial kits. Selected Sanger sequencing characterization was performed for the alleles of 11 STR markers when only one PCR primer set was available for fragment-based typing. The goal is to characterize the remaining 30 STR loci in components A–C by Sanger sequencing methods for the STR repeat regions and adjacent flanking regions. Additional characterization of the SRM is intended to support the emerging interest in next-generation sequencing technologies for forensic typing applications. Sanger methods have detected underlying polymorphisms (sequence, insertion-deletion, variation in complex motifs) typically not detected by fragment-based typing. The sequenced regions include the commercial or known PCR binding sites commonly implemented in fragment-based typing.     

A LDR‐PCR Approach for Multiplex Polymorphisms Genotyping of Severely Degraded DNA with Fragment Sizes <100 bp*

Abstract: Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini‐short tandem repeat‐polymerase chain reaction (PCR) and mini‐sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small‐scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.     

Simple and highly effective DNA extraction methods from old skeletal remains using silica columns

The recovery of DNA data from old skeletal remains is often difficult due to degraded and very low yield of extracted DNA and the presence of PCR inhibitors. Herein, we compared several silica-based DNA extraction methods from artificially degraded DNA, DNA with PCR inhibitors and DNA from old skeletal remains using quantitative real-time PCR. We present a modified large-scale silica-based extraction combined with complete demineralization, that enables maximum DNA recovery and efficient elimination of PCR inhibitors. This is performed with high concentration of EDTA solution for demineralization of bone powder followed by QIAamp^® spin columns and buffers from the QIAquick^® PCR purification kit. We have successfully used this modified technique to perform STR analysis for 55-year-old skeletal remains. The results of this study will contribute to solve the forensic cases dealing with skeletal remains.     

Forensic Identification Using a Multiplex Assay of 47 SNPs*

Abstract: As a powerful alternative to short tandem repeat (STR) profiling, we have developed a novel panel of 47 single nucleotide polymorphisms (SNPs) for DNA profiling and ABO genotyping. We selected 42 of the 47 SNPs from a panel of 86 markers that were previously validated as universal individual identification markers and identified five additional SNPs including one gender marker and four ABO loci. Match probability of the 42 validated SNPs was found to be 9.5 × 10^?18 in Han Chinese. SNP analysis correctly assessed a panel of historical cases, including both paternity identifications in trios and individual identifications. In addition, while STR profiling of degraded DNA provided information for 11 loci of 16 potential markers with low peak intensities, SNPstream^® genotyping was sufficient to identify all 47 SNPs. In summary, SNP analysis is equally effective as STR profiling, but appears more suited for individual identification than STR profiling in cases where DNA may be degraded.     

A 21‐Locus Autosomal SNP Multiplex and its Application in Forensic Science

To develop a cost‐effective technique for single‐nucleotide polymorphism (SNP) genotyping and improve the efficiency to analyze degraded DNA, we have established a novel multiplex system including 21‐locus autosomal SNPs and amelogenin locus, which was based on allele‐specific amplification (ASA) and universal reporter primers (URP). The target amplicons for each of the 21 SNPs arranged from 63 base pair (bp) to 192 bp. The system was tested in 539 samples from three ethnic groups (Han, Mongolian, and Zhuang population) in China, and the total power of discrimination (TPD) and cumulative probability of exclusion (CPE) were more than 0.99999999 and 0.98, respectively. The system was further validated with forensic samples and full profiles could be achieved from degraded DNA and 63 case‐type samples. In summary, the multiplex system offers an effective technique for individual identification of forensic samples and is much more efficient in the analysis of degraded DNA compared with standard STR typing.     

Recovery and STR amplification of DNA from RFLP membranes

Restriction fragment length polymorphism (RFLP) techniques were utilized in the forensic DNA community until the mid 1990s when less labor-intensive polymerase chain reaction short tandem repeat (PCR STR) techniques became available. During the transition from RFLP technology to PCR-based STR platforms, a method for comparing RFLP profiles to STR profiles was not developed. While the preferred approach for applying new technology to old cases would be to analyze the original biological stain, this is not always possible. For unsolved cases that previously underwent RFLP analysis, the only DNA remaining may be restriction cut and bound to nylon membranes. These studies investigate several methods for obtaining STR profiles from membrane bound DNA, including removal of bound DNA with bases, acids, detergents, various chemicals, and conventional cell extraction solutions. Direct multiplex STR amplification of template in the membrane-bound state was also explored. A partial STR profile was obtained from DNA that was recovered from an archived membrane using conventional extraction buffer components, indicating promise for recovering useful STR information from RFLP membranes that have been maintained in long-term frozen storage.     

Comparison of two whole genome amplification methods for STR genotyping of LCN and degraded DNA samples

The analysis of LCN or highly degraded DNA samples presents a challenge for forensic science. Improving the quantity and/or quality of samples would greatly increase the profiling success rate from LCN and degraded samples. Whole genome amplification (WGA) is one method that has such potential. Two commercially available WGA kits, GenomePlex and GenomiPhi, were investigated for use on LCN and degraded DNA samples. Both kits amplified genomic DNA, producing microgram quantities from sub-nanogram templates. Profiling success of LCN DNA samples was increased, with improvements of over 700% from 10pg template DNA compared to non-WGA-amplified control samples. The amplification success with degraded DNA was also improved by WGA. Degraded DNA was simulated using restriction enzymes to demonstrate that the application of WGA can result in the typing of STR loci that could not previously be amplified. An increase in artefacts, such as stutter alleles and amplification biases, were observed in many samples. Results show that WGA is capable of increasing both the quality and quantity of DNA, and has the potential to improve profiling success from difficult samples in forensic casework.