Degenerate Oligonucleotide-primed PCR Research Articles

The Comparison of Two Whole-Genome Amplification Approaches for Noninvasive Preimplantation Genetic Testing (ni-PGT) and the Application Scenario of ni-PGT during the Fresh Cycle

Recently, noninvasive preimplantation genetic testing (ni-PGT) using degenerate oligonucleotide primer PCR (DOP-PCR) and multiple annealing and looping-based amplification cycle (MALBAC)-based whole-genome amplification (WGA) methods has demonstrated predictable results in embryo testing. However, a considerable heterogeneity of results has been reported in numerous studies on these two WGA methods. Our aim was to evaluate the current WGA method for ni-PGT while further clarifying the applicable scenarios of ni-PGT in the fresh cycle. A total of 173 embryos were tested with trophectoderm biopsy and ni-PGT. In the whole preimplantation genetic testing, the clinical concordance rates of the detection results of DOP-PCR and MALBAC with the corresponding trophectoderm biopsy results were 64.12% (84/131) and 68.99% (89/129), respectively (P=0.405). However, in the detection of abnormal embryos, the detection efficiency of ni-PGT is significantly improved [MALBAC: 96.55% versus 68.99% (P<0.001); and DOP-PCR: 89.09% versus 64.12% (P<0.001)]. In addition, the diagnostic efficiency of ni-PGT in low-quality blastocysts was significantly higher than that in high-quality blastocysts [MALBAC: 95.24% versus 51.85% (P=0.001); and DOP-PCR: 91.30% versus 48.15% (P=0.001)]. These results contribute to further understanding ni-PGT and to clarifying its application scenario in the fresh cycle.

Engineering the Metabolic Profile of Clostridium cellulolyticum with Genomic DNA Libraries

Clostridium cellulolyticum H10 (ATCC 35319) has the ability to ferment cellulosic substrates into ethanol and weak acids. The growth and alcohol production rates of the wild-type organism are low and, therefore, targets of metabolic engineering. A genomic DNA expression library was produced by a novel application of degenerate oligonucleotide primed PCR (DOP-PCR) and was serially enriched in C. cellulolyticum grown on cellobiose in effort to produce fast-growing and productive strains. The DNA library produced from DOP-PCR contained gene-sized DNA fragments from the C. cellulolyticum genome and from the metagenome of a stream bank soil sample. The resulting enrichment yielded a conserved phage structural protein fragment (part of Ccel_2823) from the C. cellulolyticum genome that, when overexpressed alone, enabled the organism to increase the ethanol yield by 250% compared to the plasmid control strain. The engineered strain showed a reduced production of lactate and a 250% increased yield of secreted pyruvate. Significant changes in growth rate were not seen in this engineered strain, and it is possible that the enriched protein fragment may be combined with the existing rational metabolic engineering strategies to yield further high-performing cellulolytic strains.

Mapping Replication Timing in Single Mammalian Cells.

Replication timing (RT) is the temporal order in which genomic DNA is replicated during S phase. Early and late replication correlate with transcriptionally active and inactive chromatin compartments, but mechanistic links between large-scale chromosome structure, transcription, and replication are still enigmatic. A proper RT program is necessary to maintain the global epigenome that defines cell identity, suggesting that RT is critical for epigenome integrity by facilitating the assembly of different types of chromatin at different times during S phase. RT is regulated during development and has been found to be altered in disease. Thus, RT can identify stable epigenetic differences distinguishing cell types, and can be used to help stratify patient outcomes and identify markers of disease. Most methods to profile RT require thousands of S-phase cells. In cases where cells are rare (e.g., early-stage embryos or rare primary cell types) or consist of a heterogeneous mixture of cell states (e.g., differentiation intermediates), or when the interest is in determining the degree of stable epigenetic heterogeneity within a population of cells, single-cell measurements of RT are necessary. We have previously developed single cell Repli-seq, a method to measure replication timing in single cells using DNA copy number quantification. To date, however, single-cell Repli-seq suffers from relatively low throughput and high costs. Here, we describe an improved single-cell Repli-seq protocol that uses degenerate oligonucleotide-primed PCR (DOP-PCR) for uniform whole-genome amplification and uniquely barcoded primers that permit early pooling of single-cell samples into a single library preparation. We also provide a bioinformatics platform for analysis of the data. The improved throughput and decreased costs of this method relative to previously published single-cell Repli-seq protocols should make it considerably more accessible to a broad range of investigators. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Whole Genome Amplification (WGA) of single cells and sequence library construction. Basic Protocol 2: Deriving and displaying single-cell replication timing data from whole genome sequencing.

Cloning and physical localization of male-biased repetitive DNA sequences in Spinacia oleracea (Amaranthaceae)

Spinach (Spinacia oleracea Linnaeus, 1753) is an ideal material for studying molecular mechanisms of early-stage sex chromosome evolution in dioecious plants. Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) technique facilitates the retrotransposon-relevant studies by enriching specific repetitive DNA sequences from a micro-dissected single chromosome. We conducted genomic subtractive hybridization to screen sex-biased DNA sequences by using the DOP-PCR amplification products of micro-dissected spinach Y chromosome. The screening yielded 55 male-biased DNA sequences with 30 576 bp in length, of which, 32 DNA sequences (12 049 bp) contained repeat DNA sequences, including LTR/Copia, LTR/Gypsy, simple repeats, and DNA/CMC-EnSpm. Among these repetitive DNA sequences, four DNA sequences that contained a fragment of Ty3-gypsy retrotransposons (SP73, SP75, SP76, and SP77) were selected as fluorescence probes to hybridization on male and female spinach karyotypes. Fluorescence in situ hybridization (FISH) signals of SP73 and SP75 were captured mostly on the centromeres and their surrounding area for each homolog. Hybridization signals primarily appeared near the putative centromeres for each homologous chromosome pair by using SP76 and SP77 probes for FISH, and sporadic signals existed on the long arms. Results can be served as a basis to study the function of repetitive DNA sequences in sex chromosome evolution in spinach.

Cloning and physical localization of male-biased repetitive DNA sequences in Spinacia oleracea (Amaranthaceae).

Spinach (Spinacia oleracea Linnaeus, 1753) is an ideal material for studying molecular mechanisms of early-stage sex chromosome evolution in dioecious plants. Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) technique facilitates the retrotransposon-relevant studies by enriching specific repetitive DNA sequences from a micro-dissected single chromosome. We conducted genomic subtractive hybridization to screen sex-biased DNA sequences by using the DOP-PCR amplification products of micro-dissected spinach Y chromosome. The screening yielded 55 male-biased DNA sequences with 30 576 bp in length, of which, 32 DNA sequences (12 049 bp) contained repeat DNA sequences, including LTR/Copia, LTR/Gypsy, simple repeats, and DNA/CMC-EnSpm. Among these repetitive DNA sequences, four DNA sequences that contained a fragment of Ty3-gypsy retrotransposons (SP73, SP75, SP76, and SP77) were selected as fluorescence probes to hybridization on male and female spinach karyotypes. Fluorescence in situ hybridization (FISH) signals of SP73 and SP75 were captured mostly on the centromeres and their surrounding area for each homolog. Hybridization signals primarily appeared near the putative centromeres for each homologous chromosome pair by using SP76 and SP77 probes for FISH, and sporadic signals existed on the long arms. Results can be served as a basis to study the function of repetitive DNA sequences in sex chromosome evolution in spinach.

Plant Chromosome-Specific Probes by Microdissection of a Single Chromosome: Is That a Reality?

Painting plant chromosomes through chromosomal in situ suppression (CISS) hybridization has long been considered impracticable. Seeking to build specific and complex probes from a single microdissected chromosome, we employed human chromosomes as models to standardize all the necessary steps for application in plants. Human metaphases were used to define the adequate conditions for microdissection, chromosome DNA amplification and labeling through degenerate oligonucleotide-primed PCR, and in situ hybridization stringency. Subsequently, these methodologies were applied in the plant species Zea mays (chromosome 1) and Capsicum annuum (chromosome 7 or 8). The high quality of human and plant cytogenetic preparations and the meticulous standardization of each step, especially the most critical ones – microdissection and first round of DNA amplification – were crucial to eliminate the signs of non-specific hybridization and for direct application in plants. By overcoming these challenges, we obtained chromosome-specific probes, which allowed to achieve a clear and uniform painting of the entire target chromosomes with little or no background, evidencing their complexity and specificity. Despite the high amount of ubiquitous repetitive sequences in plant genomes, the main drawback for chromosome painting, we successfully employed our methodology on two plant species. Both have more than 80% repetitive sequences, which is compared to the human genome (66–69%). This is the first time that plant chromosome-specific probes were successfully obtained from a single A mitotic or meiotic microdissected chromosome. Thereby, we assume that chromosome painting through microdissection and CISS hybridization can now be considered a reality in the field of plant cytogenetics.

Use of degenerate oligonucleotide primed polymerase chain reaction for detection of chicken anaemia virus contamination in avian viral vaccines

For quality control of biologicals of veterinary use, the absence of extraneous agents needs to be certified. One of the requirements for quality control of avian viral vaccines is to demonstrate freedom from extraneous and adventitious pathogenic agents, like chicken anaemia virus (CAV). In this study, a degenerate oligonucleotide primed PCR (DOP-PCR) for the detection of CAV was developed. Degenerate oligonucleotide primers were selected based on sequences corresponding to conserved regions of VP1 gene. After spiking of CAV genomic DNA to an infectious laryngotracheitis virus (ILTV) vaccine, detection limit for the test was 3.056×10-9 ng/µl. To evaluate the performance of the test, 11 avian viral vaccines including infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and ILTV vaccines from 5 manufacturers were screened for CAV and no contamination was detected. The test described here may provide a rapid, sensitive and specific method for contamination detection of avian viral vaccines with CAV, and may be applied for quality control of live and killed commercial vaccines.

Genetic analysis by chromosome sorting and painting: phylogenetic and diagnostic applications.

Chromosome sorting from fluid suspensions of metaphase chromosomes using a fluorescence-activated cell sorter has been used for a number of years to produce chromosome-specific genomic libraries and other reagents for chromosome mapping. Improved techniques for fluorescence in situ hybridisation and the amplification and labelling of sorted chromosomes using degenerate oligonucleotide-primed PCR have led to the widespread use of chromosome painting both for the resolution of complex chromosome aberrations and for the study of karyotype evolution by cross-species reciprocal chromosome painting. The chromosomes of a large number of different species have been sorted and used to make chromosome-specific paints and already new data challenging results of earlier phylogenetic studies have been obtained. Sorted chromosomes provide the resource for multicolour chromosome analysis of all chromosomes simultaneously. Such reagents are now available for all human and mouse chromosomes and are proving particularly useful in the analysis of cancer chromosomes.

Preparing Single-cell DNA Library Using Nextera for Detection of CNV.

Single-cell DNA sequencing is a powerful tool to evaluate the state of heterogeneity of heterogeneous tissues like cancer in a quantitative manner that bulk sequencing can never achieve. DOP-PCR (Degenerate Oligonucleotide-Primed Polymerase Chain Reaction), MDA (Multiple Displacement Amplification), MALBAC (Multiple Annealing and Looping-Based Amplification Cycles), LIANTI (Linear Amplification via Transposon Insertion) and TnBC (Transposon Barcoded) have been the primary choices to prepare single-cell libraries. TnBC library prep method is a simple and versatile methodology, to detect copy number variations or to obtain the absolute copy numbers of genes per cell.

Genetic evaluation of bovine papillomavirus types detected in equine sarcoids in Poland.

Equine sarcoids are the most common neoplasms in horses. Bovine papilloma- virus type 1 (BPV-1) is the main viral type identified in equine sarcoids in Europe. The aim of the present study was to genetically evaluate BPV types based on DNA analyses of the CDS of the L1 gene. The presence of BPV DNA was confirmed by Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP PCR) with FAP59/FAP64 consensus primers. The DNA was detected in 21/40 (52.5%) of clinically diagnosed sarcoids. More than half of 14 isolates (66.7%) shared 100% homology with BPV-1 Deltapapillomavirus 4 isolate 09 asi UK (Acc. No. MF384289) and 99% nucleotide identity with BPV-1 isolate EqSarc1 (Acc. No. JX678969). A comparison with BPV-1 isolate EqSarc1 revealed one silent mutation in C5827T which did not change the aminoacid codon. The remaining 6 isolates (28.6%) shared 100% nucleotide identity with the BPV-1 (Acc. No. X02346) "wild type" isolate, and 1 isolate (4.8%) demonstrated 99% nucleotide identity with BPV-2 (Acc. No. M20219). Variants of BPV-1 isolate EqSarc1 (Acc. No. JX678969) constitute the most prevalent type of BPV-1 in Polish horses.

Assessment of DNA degradation of buccal cells under humid conditions and DNA repair by DOP-PCR using locked nucleic acids.

We analyzed the degradation level of DNA from buccal cells under humid conditions using quantitative PCR analysis. Gauze samples with buccal cells were incubated for up to 12 months under three different conditions (25 °C/dry, 25 °C/humid, or 40 °C/humid). The degradation was evaluated based on two degradation ratios (129:41 and 305:41 bp). DNA degraded slowly under the 25 °C/humid condition, and significant differences in the two degradation ratios were detected between 25 °C/dry and 25 °C/humid conditions after 12 months. Moreover, the degradation rapidly progressed under the 40 °C/humid condition, and the two degradation ratios in this condition were much lower than those from 25 °C/dry and 25 °C/humid conditions after a short incubation period (3 months). To evaluate the effect of DNA repair on low-copy degraded DNA, degenerate oligonucleotide-primed PCR (DOP-PCR) was performed before short tandem repeats (STR) genotyping. As a standard DOP-PCR, we used a 22-base primer with 10 degenerate sequences (5'-CTCGAGNNNNNNNNNNATGTGG-3'), and additionally designed DOP-PCR primers with 2, 4, 6, or 8 locked nucleic acids (LNAs). When slightly degraded DNA (305:41-bp ratio = 0.60) was used, DOP-PCR significantly increased the fluorescent intensity and success rate of genotyping using Identifiler and Globalfiler kits. In particular, the reaction with four LNAs produced the highest value. However, such benefits were not observed in the analysis of moderately degraded DNA (305:41-bp ratio = 0.13). Although the recovery rates of STR profiles by DOP-PCR were dependent on the degradation level of low-copy DNA, the effectiveness of DOP-PCR highlights the potential of LNA for degenerate sequences.

Comparison of Amplification Methods of Single Trophoblast Cells for Their Subsequent Analysis Using Metaphase Comparative Genomic Hybridization

Single trophoblast cells circulating in the bloodstream of pregnant women are potential objects for noninvasive prenatal diagnosis. Owing to the very low concentration of cells of a fetal nature in the peripheral maternal blood, the choice of the method for whole genome amplification of the genetic material becomes topical. The key point in the use of single cells of a fetal nature for noninvasive prenatal diagnosis is to obtain DNA in an amount and of a quality acceptable for the analysis. In order to select the optimal method for whole genome amplification, a model experiment was conducted. We compared three different methods of whole genome amplification: linker-adaptor polymerase chain reaction (LA-PCR), degenerate oligonucleotide- primed PCR (DOP-PCR), and multiple displacement amplification (MDA). Subsequent analysis of the amplification products was performed by metaphase comparative genomic hybridization in order to evaluate the molecular karyotype of cells of a fetal nature with the known chromosome complement. As a result, an optimal method for whole genome amplification of the genetic material of single cells in a model experiment was determined by linker-adaptor PCR, which showed a more uniform representation of the genome regions compared with the other methods used.

Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques

Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.

Improved DOP-PCR (iDOP-PCR): A robust and simple WGA method for efficient amplification of low copy number genomic DNA.

Whole-genome amplification (WGA) techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR), which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR). We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase) with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.

Isolation and application of P genome-specific DNA sequences of Agropyron Gaertn. in Triticeae.

Different types of P genome sequences and markers were developed, which could be used to analyze the evolution of P genome in Triticeae and identify precisely wheat- A. cristatum introgression lines. P genome of Agropyron Gaertn. plays an important role in Triticeae and could provide many desirable genes conferring high yield, disease resistance, and stress tolerance for wheat genetic improvement. Therefore, it is significant to develop specific sequences and functional markers of P genome. In this study, 126 sequences were isolated from the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) products of microdissected chromosome 6PS. Forty-eight sequences were identified as P genome-specific sequences by dot-blot hybridization and DNA sequences analysis. Among these sequences, 22 displayed the characteristics of retrotransposons, nine and one displayed the characteristics of DNA transposons and tandem repetitive sequence, respectively. Fourteen of 48 sequences were determined to distribute on different regions of P genome chromosomes by fluorescence in situ hybridization, and the distributing regions were as following: all over P genome chromosomes, centromeres, pericentromeric regions, distal regions, and terminal regions. We compared the P genome sequences with other genome sequences of Triticeae and found that the similar sequences of the P genome sequences were widespread in Triticeae, but differentiation occurred to various extents. Additionally, thirty-four molecular markers were developed from the P genome sequences, which could be used for analyzing the evolutionary relationship among 16 genomes of 18 species in Triticeae and identifying P genome chromatin in wheat-A. cristatum introgression lines. These results will not only facilitate the study of structure and evolution of P genome chromosomes, but also provide a rapid detecting tool for effective utilization of desirable genes of P genome in wheat improvement.

Chromosome sorting for the masses?

Chromosome sorting for the masses?

Raman spectroscopy detects phenotypic differences among Escherichia coli enriched for 1-butanol tolerance using a metagenomic DNA library.

Advances in Raman spectroscopy are enabling more comprehensive measurement of microbial cell chemical composition. Advantages include results returned in near real-time and minimal sample preparation. In this research, Raman spectroscopy is used to analyze E. coli with engineered solvent tolerance, which is a multi-genic trait associated with complex and uncharacterized phenotypes that are of value to industrial microbiology. To generate solvent tolerant phenotypes, E. coli transformed with DNA libraries are serially enriched in the presence of 0.9% (v/v) and 1.1% (v/v) 1-butanol. DNA libraries are created using degenerate oligonucleotide primed PCR (DOP-PCR) from the genomic DNA of E. coli, Clostridium acetobutylicum ATCC 824, and the metagenome of a stream bank soil sample, which contained DNA from 72 different phyla. DOP-PCR enabled high efficiency library cloning (with no DNA shearing or end-polishing) and the inclusion un-culturable organisms. Nine strains with improved tolerance are analyzed by Raman spectroscopy and vastly different solvent-tolerant phenotypes are characterized. Common among these are improved membrane rigidity from increasing the fraction of unsaturated fatty acids at the expense of cyclopropane fatty acids. Raman spectroscopy offers the ability to monitor cell phenotype changes in near real-time and is adaptable to high-throughput screening, making it relevant to metabolic engineering.

Effects of salinity on activity and expression of enzymes involved in ionic, osmotic, and antioxidant responses in Eurya emarginata

Major constraints imposed by salinity, include ionic, osmotic, and oxidative stresses, which significantly affect plant growth. In an attempt to explore the mechanisms underlying salinity tolerance, enzymatic activity, and expression of genes involved in ionic, osmotic, and antioxidant responses of the typical coastal plant Eurya emarginata were examined under stressed conditions of 150 and 300 mM NaCl for 35 days. Physiological and biochemical parameters were also assessed. Salt stress adversely affected the growth of E.emarginata, as evidenced by reduced fresh weight (FW), leaf water content (LWC), and chlorophyll a and b contents. The inhibition of growth could be attributed to an increase in malondialdehyde (MDA) and proline contents, and Na+ concentration, and a decrease in K+ uptake in the leaves of E.emarginata. MDA and proline contents, and Na+ and K+ concentrations were closely related to activities of H+-ATPase, pyrroline-5-carboxylate synthetase (P5CS), and proline dehydrogenase (PDH), and antioxidant enzymes such as Cu, Zn superoxide dismutase (Cu/Zn-SOD), catalase (CAT), and glutathione peroxidase (GPx). To understand the basis for the observed differences, five genes encoding these enzymes were cloned from the leaves of E.emarginata by degenerate oligonucleotide-primed PCR to assess transcript levels under salt stress. At 150 mM NaCl, mRNA levels of CAT, GPx, P5CS, and H+-ATPase sharply increased, whereas mRNA levels of Cu/Zn-SOD, CAT, GPx, H+-ATPase, and PDH were significantly down-regulated at 300 mM NaCl. Therefore, the cloned genes may play an important role in determining how salinity tolerance is conferred in E. emarginata, which will facilitate further studies on the regulation of salt stress at the molecular level for this species.

Rapid cloning and bioinformatic analysis of spinach Y chromosome-specific EST sequences.

The genome of spinach single chromosome complement is about 1000 Mbp, which is the model material to study the molecular mechanisms of plant sex differentiation. The cytological study showed that the biggest spinach chromosome (chromosome 1) was taken as spinach sex chromosome. It had three alleles of sex-related X,X(m) and Y. Many researchers have been trying to clone the sex-determining genes and investigated the molecular mechanism of spinach sex differentiation. However,there are no successful cloned reports about these genes. A new technology combining chromosome microdissection with hybridization-specific amplification (HSA) was adopted. The spinach Y chromosome degenerate oligonucleotide primed-PCR (DOP-PCR) products were hybridized with cDNA of the male spinach flowers in florescence. The female spinach genome was taken as blocker and cDNA library specifically expressed in Y chromosome was constructed. Moreover, expressed sequence tag (EST) sequences in cDNA library were cloned, sequenced and bioinformatics was analysed. There were 63 valid EST sequences obtained in this study. The fragment size was between 53 and 486 bp. BLASTn homologous alignment indicated that 12 EST sequences had homologous sequences of nucleic acids, the rest were new sequences. BLASTx homologous alignment indicated that 16 EST sequences had homologous protein-encoding nucleic acid sequence. The spinach Y chromosome-specific EST sequences laid the foundation for cloning the functional genes, specifically expressed in spinach Y chromosome. Meanwhile, the establishment of the technology system in the research provided a reference for rapid cloning of other biological sex chromosome-specific EST sequences.

Modified DOP-PCR for improved STR typing of degraded DNA from human skeletal remains and bloodstains.

Forensic and ancient DNA samples often are damaged and in limited quantity as a result of exposure to harsh environments and the passage of time. Several strategies have been proposed to address the challenges posed by degraded and low copy templates, including a PCR based whole genome amplification method called degenerate oligonucleotide-primed PCR (DOP-PCR). This study assessed the efficacy of four modified versions of the original DOP-PCR primer that retain at least a portion of the 5' defined sequence and alter the number of bases on the 3' end. The use of each of the four modified primers resulted in improved STR profiles from environmentally-damaged bloodstains, contemporary human skeletal remains, American Civil War era bone samples, and skeletal remains of WWII soldiers over those obtained by previously described DOP-PCR methods and routine STR typing. Additionally, the modified DOP-PCR procedure allows for a larger volume of DNA extract to be used, reducing the need to concentrate the sample and thus mitigating the effects of concurrent concentration of inhibitors.