Amplification Yield Research Articles

Searching for alternative high DNA-yielding bone types for DNA analysis of aged skeletal remains

The petrous bone contains significantly higher amounts of DNA than any other human bone. Because of highly destructive sampling and because it is not always part of the recovered remains, the need for alternative sources of DNA is important. To identify additional optimal bone types, petrous bones were compared to femurs, tali, and calcanei sampled from 66 adult skeletons from two distinct modern-era Christian cemeteries. An extraction method employing full demineralization was used to obtain DNA, real-time PCR quantification to ascertain DNA quantity and degradation, and a commercial forensic short tandem repeats (STR) PCR amplification kit to determine genetic profiles. Statistical analysis was performed to explore the differences in DNA yield, DNA degradation, and success of STR amplification. A systematic studies exploring intra-skeletal variability in DNA preservation including various excavation sites differing by time period and geographical position are rare, and the second part of the investigation was based on a comparison of both archaeological sites, which allowed us to compare the effect of different post-mortem intervals and environmental conditions on DNA preservation. The older burial site in Črnomelj was active between the 13th and 18th century, whereas the more recent Polje burial was in use from the 16th to 19th century, creating different temporal and geographical environments. Results for the Črnomelj burial site revealed that the petrous bone outperformed all other bone types studied, except the calcaneus. At the Polje archeological site calcanei, tali, and femurs yielded the same STR typing success as petrous bones. The results obtained highlight the importance of careful bone sample selection for DNA analysis of aged skeletal remains. In addition to petrous bones, calcanei were found to be an alternative source of DNA when older burial sites are investigated. When more recent burial sites are processed, calcanei, tali, and femurs should be sampled besides petrous bones, not only because they exhibited good performance, but also because of easier sampling and easier grinding in the case of trabecular bones. This study contributes valuable insights into the potential use of various skeletal types as a source of DNA for investigation of aged skeletal remains, and it offers practical implications for forensic and archaeological investigations.

Utilization of Modified Touchdown qPCR to Enhance Sensitivity and Specificity for Genes with Restricted Expression and Low Optimum Temperature Primers

Abstract Background: Quantitative polymerase chain reaction (qPCR) is one of the most used methods to analyze gene expression. However, if the annealing temperature in the qPCR cycle is lower than 55°C, nonspecific amplification may occur. On the other hand, some sets of primers can only function optimally at the annealing temperature below 55°C. Thus, a protocol allowing both specific amplification and large yield in low annealing temperature is needed. Methods: qPCR on the target ribosomal protein L13a (rpl13a) and natriuretic peptide A (nppa) from samples of zebrafish embryos and larvae was performed in triplicates using three different protocols: routine qPCR, gradient qPCR, and modified touchdown qPCR. The cycle of quantification (Cq) value and melt peak data were compared between the routine or gradient qPCR and the modified touchdown qPCR. Results: For rpl13a that was adequately and specifically amplified by both routine and touchdown qPCR, modified touchdown qPCR decreased the average Cq by 3.68–4.66 (P < 0.05). For nppa, a gene with restricted expression that could only be amplified substantially but nonspecifically at the annealing temperature of 50°C, modified touchdown qPCR increased the amplification specificity by forming only one melt peak during melt curve analysis and yielding a substantial amount of amplicon with the Cq of 22.48–22.51. Conclusions: The modified touchdown qPCR with low final annealing temperature was able to produce specific amplification with better yield and detection sensitivity. This method is especially useful for primer sets with low optimum annealing temperature.

A sensitive one-pot ROA assay for rapid miRNA detection

MicroRNAs (miRNAs) and short RNA fragments (18–25 nt) are crucial biomarkers in biological research and disease diagnostics. However, their accurate and rapid detection remains a challenge, largely due to their low abundance, short length, and sequence similarities. In this study, we report on a highly sensitive, one-step RNA O-circle amplification (ROA) assay for rapid and accurate miRNA detection. The ROA assay commences with the hybridization of a circular probe with the test RNA, followed by a linear rolling circle amplification (RCA) using dUTP. This amplification process is facilitated by U-nick reactions, which lead to an exponential amplification for readout. Under optimized conditions, assays can be completed within an hour, producing an amplification yield up to the microgram level, with a detection limit as low as 0.15 fmol (6 pM). Notably, the ROA assay requires only one step, and the results can be easily read visually, making it user-friendly. This ROA assay has proven effective in detecting various miRNAs and phage ssRNA. Overall, the ROA assay offers a user-friendly, rapid, and accurate solution for miRNA detection.

Direct TAMRA-dUTP labeling of M. tuberculosis genes using loop-mediated isothermal amplification (LAMP)

Fluorescent molecule-based direct labeling of amplified DNA is a sensitive method employed across diverse DNA detection and diagnostics systems. However, using pre-labeled primers only allows for the attachment of a single fluorophore to each DNA strand and any modifications of the system are less flexible, requiring new sets of primers. As an alternative, direct labeling of amplified products with modified nucleotides is available, but still poorly characterized. To address these limitations, we sought a direct and adaptable approach to label amplicons produced through Loop-mediated isothermal amplification (LAMP), using labeled nucleotides (dUTPs) rather than primers. The focus of this study was the development and examination of a direct labeling technique of specific genes, including those associated with drug resistance in Mycobacterium tuberculosis. We used 5-(3-Aminoallyl)-2′-deoxyuridine-5′triphosphate, tagged with 5/6-TAMRA (TAMRA-dUTP) for labeling LAMP amplicons during the amplification process and characterized amplification and incorporation efficiency. The optimal TAMRA-dUTP concentration was first determined based on amplification efficiency (0.5% to total dNTPs). Higher concentrations of modified nucleotides reduced or completely inhibited the amplification yield. Target size also showed to be determinant to the success of amplification, as longer sequences showed lower amplification rates, thus less TAMRA incorporated amplicons. Finally, we were able to successfully amplify all four M. tuberculosis target genes using LAMP and TAMRA-modified dUTPs.

C2CAplus: A One-Pot Isothermal Circle-to-Circle DNA Amplification System.

Rolling circle amplification (RCA) is a widely used DNA amplification method that uses circular template DNA as input and produces multimeric, linear single- or double-stranded DNA. Circle-to-circle amplification (C2CA) has further expanded this method by implementing product recircularization using restriction and ligation, leading to a higher amplification yield and enabling the generation of circular products. However, C2CA is a multistep, nonisothermal method, requiring multiple fluid manipulations and thereby compromises several advantages of RCA. Here, we improved C2CA to implement a one-pot, single step, isothermal reaction at temperatures ranging from 25 to 37 °C. Our C2CAplus method is simple, robust, and produces large quantities of product DNA that can be seen with the naked eye.

Enhancement of fungal DNA templatesand PCR amplification yield by three types of nanoparticles

Enhancement of fungal DNA templatesand PCR amplification yield by three types of nanoparticles

(Digital Presentation) An Electrochemical Sensor in a Disc for Real-Time PCR Assisted with Thermal Convective Flow

Multiple microfluidic devices incorporating real-time polymerase chain reaction (PCR) for the quantification of DNA targets have been reported. They show features such as fast analysis time, reduced reagent consumption, and portability. Integration of real-time PCR in a centrifugal microfluidic disc has been also proposed due to the ease of automation and parallelization of microfluidic operations. Previously, it has been demonstrated the implementation of thermal convective PCR (TC-PCR) in a CD using a ring-structured microchannel. In this case, the microchannel is supplied with the temperature necessary to perform the complete thermal cycle: Denaturation, annealing, and extension. The flow generated by thermal convection, in combination with the centrifugal force moves the sample through the entire microchannel due to the difference in densities. In this work, we present the implementation in a CD of an electrochemical sensor combined with TC-PCR to quantify the amplification yield in real-time. We present advances related to the calibration of the electrochemical sensor, and the implementation of the TC-PCR in a CD as well as a preliminary simulation in COMSOL.

Recombinase polymerase amplification in minimally buffered conditions

Application of recombinase polymerase amplification (RPA) for pH-based detection of DNA amplification has been investigated. Commercial RPA kits from TwistDx are modified to minimize their pH buffering capacity. Due to the RPA's unique biochemistry, removal of tris from the amplification kit is not enough to lower the buffering capacity of the RPA assay. Even in the absence of tris, RPA components in the commercial kit intrinsically buffer the pH. We show different strategies to minimize the buffering capacity of the RPA kit, while maintaining the amplification efficiency. Even in minimally buffered conditions, it is noticed that RPA's amplification yield is not high enough to overcome the assay's intrinsic buffering capacity. The effect of pyrophosphate precipitation in RPA on the reaction's pH have also been addressed. In conclusion, this work highlights strategies and considerations for the development of pH-based assays from nucleic acid amplification methods which involve ancillary enzymes that catalyze nucleotide hydrolysis.

Design of Oligonucleotides for Allele-Specific Amplification Based on PCR and Isothermal Techniques.

Single-nucleotide variations have been associated to various genetic diseases, variations on drug efficiency, and differences in cancer prognostics. The detection of these changes in nucleic acid sequences from patient samples is particularly useful for accurate diagnosis, therapeutics, and disease management. A reliable allele-specific amplification is still an important challenge for molecular-based diagnostic technologies. In the last years, allele-specific primers have been designed for promoting the enrichment of certain variants, based on a higher stability of primer/template duplexes. Also, several methods are based on the addition of a blocking oligonucleotide that prevent the amplification of a specific variant, enabling that other DNA variants can be observed. In this context, genotyping methods based on isothermal amplification techniques are increasing, especially those assays aimed to be deployed at point-of-care applications. The correct selection of target sequences is crucial for reaching the required analytical performances, in terms of reaction time, amplification yield, and selectivity. The present chapter describes the design criteria for the selection of primers and blockers for relevant PCR approaches and novel isothermal strategies. Several successful examples are provided in order to highlight the main design restrictions and the potential to be extended to other applications.

The disc-shaped microcarriers: A new tool for increasing harvesting of adipose-derived mesenchymal stromal cells

Human adipose-derived mesenchymal stromal cells (A-MSC) are promising tools for regenerative medicine, but their in vitro amplification before administration is still an issue. Microcarriers emerge as a novel approach for A-MSC expansion in bioreactors. Here, microcarriers with different shapes and functionalizations were compared according to their efficiency for A-MSC expansion. In particular, novel disc shape microcarriers were compared to the commonly used spherical microcarriers. This geometry increases their specific surface while presenting a flat surface, close to monolayer culture conditions. Several parameters were evaluated, such as cell amplification and harvesting yields, cell viability, and cell identity after culture. We observed that microcarriers shape and functionalization impact several A-MSC amplification process steps. However, GhaterDisc-3 microcarriers were the best alternative since harvesting is highly facilitated compared to other microcarriers with a yield of virtually 100 %. Furthermore, A-MSC identity was maintained, since A-MSC keep their characteristic immunophenotype and tri-lineage differentiation ability. Moreover, as these microcarriers are free from any animal derived components, they might be suitable for a use in a GMP compliant large-scale A-MSC production for biomedical applications.

Evaluating the impact of sampling strategies and bioinformatics on ethanol-based DNA metabarcoding

Recent developments on ethanol-based DNA (etDNA) metabarcoding have shown that it is possible to extract meaningful information about macroinvertebrate community diversity and composition from the ethanol used to preserve bulk samples. The major advantages of this molecular approach are the reduced processing time and costs, and the possibility to keep specimens intact for other experiments. Yet, organisms with highly sclerotised exoskeleton or that are rare in the sample have been found to release a lower amount of DNA into solution and tend to be consistently missed by etDNA metabarcoding, thereby compromising the viability of the method. Few studies have shown that the first steps of the metabarcoding workflow are crucial for the good performance of etDNA-based assays, such as the decision on storage time before sampling and the ethanol phase to be analysed, the inclusion of pre-treatment strategies (i.e., freezing), and the choice of the DNA extraction protocol. In this study, we aimed to evaluate the combined effect of various technical choices on the performance of etDNA metabarcoding, considering factors such as sample volume, ethanol phase of sorted and unsorted samples, pre-capture treatments (evaporation vs filtration) and bioinformatic pipelines. Through the application of decision-tree models, our preliminary data revealed that the increase of volume (by itself) is enough to improve PCR amplification yields and proportion of families matching the morphological identifications, with great impact on the detection of hard-bodied and cased taxa. Also, no major differences among phases with or without a sorting step nor among bioinformatic pipelines were detected, particularly at higher volumes. Our results suggest that the higher performance (with lower observed variation) in taxonomic detection at higher volumes is likely a consequence of a higher availability of longer fragments of DNA in solution. This study highlights the importance of understanding the impact of technical choices to improve the efficiency of a DNA-based method, and reinstates etDNA metabarcoding as a potential method in the context of biomonitoring.

DMSO Improves the Ski-Slope Effect in Direct PCR

Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes.

Optimization of on-bead emulsion polymerase chain reaction based on single particle analysis

Emulsion polymerase chain reaction (ePCR) enables parallel amplification of millions of different DNA molecules while avoiding bias and chimeric byproducts, essential criteria for applications including next generation sequencing, aptamer selection, and protein-DNA interaction studies. Despite these advantages, ePCR remains underused due to the lack of optimal starting conditions, straightforward methods to evaluate success, and guidelines for tuning the reaction. This knowledge has been elusive for bulk emulsion generation methods, such as stirring and vortexing, the only methods that can emulsify libraries of ≥108 sequences within minutes, because these emulsions have not been characterized in ways that preserve the heterogeneity that defines successful ePCR. Our study quantifies the outcome of ePCR from conditions specified in the literature using single particle analysis, which preserves this heterogeneity. We combine ePCR with magnetic microbeads and quantify the amplification yield via qPCR and the proportion of clonal and saturated beads via flow cytometry. Our single particle level analysis of thousands of beads resolves two key criteria that define the success of ePCR: 1) whether the target fraction of 20% clonal beads predicted by the Poisson distribution is achieved, and 2) whether those beads are partially or maximally covered by amplified DNA. We found that among the two concentrations of polymerase tested, only the higher one, which is 20-fold more than the concentration recommended for conventional PCR, could yield sufficient PCR products. Dramatic increases in the concentrations of reverse primer and nucleotides recommended in literature gave no measurable change in outcome. We thus provide evidence-based starting conditions for effective and economical ePCR for real DNA libraries and a straightforward workflow for evaluating the success of tuning ePCR prior to downstream applications.

Enhancement of PCR efficiency using mono- and disaccharides

Polymerase chain reaction is the most commonly used approach for nucleic acids amplification. Despite the variety of PCR methods have been proposed, new techniques are being developed to improve this reaction. We found that, in general, mono- and disaccharides can serve as effective PCR enhancers. Unlike oligo- and polysaccharides, low molecular-weight carbohydrates accelerate amplification and increase products yield. The ability of carbohydrates to enhance PCR is not related to their reducing property. The best result was obtained for sucrose providing the most specific and reliable amplification. The effect of carbohydrates is leveled as the size of the amplification region increases.

Comparison between cryobiopsy and forceps biopsy in detection of epidermal growth factor receptor amplification in non-small-cell lung cancer

BackgroundNon-small-cell lung cancer (NSCLC) represents 85% of lung cancer cases. Genotyping is now considered as a cornerstone in proper management and better results of such cancers, especially with targeted therapy. Cryobiopsy is a promising tool in NSCLC to obtain larger samples, with well-preserved tissue sufficient for accurate histopathological and gene detection.AimTo compare cryobiopsy and ordinary forceps results in detection of epidermal growth factor receptor (EGFR) amplification in NSCLC.Materials and methodsSamples from 34 patients with proven NSCLC by cryobiopsy versus forceps biopsy were compared for size, quality, and diagnostic yield of EGFR gene amplification.ResultsThe samples obtained by cryoprobe had larger size and better artifact-free areas with more diagnostic yield of EGFR gene amplification (29.4%) versus with forceps biopsy (8.8%), with gene amplification showing higher statistical significance in younger patients, never smokers, and women (P<0.001).ConclusionCryobiopsy is an excellent tool for larger, better-quality sampling and for higher diagnostic yield of EGFR amplification in NSCLC.

An Alternative Hot Start PCR Method Using a Nuclease-Deficient ExoIII from Escherichia coli.

The Hot Start polymerase chain reaction (Hot Start PCR) is designed to reduce off-target amplification by blocking DNA polymerase extension at room temperature until the desired temperature is reached. In this study, we investigated a new method of Hot Start PCR that uses a modified Escherichia coli Exonuclease III (EcoExoIIIM) by substituting residues in the DNA-binding pocket and catalytic center. The results showed that PCR amplification yield and specificity were significantly promoted by the addition of EcoExoIIIM. We hypothesize that non-specific binding of primers at room temperature is prevented by binding of the primed template by EcoExoIIIM, which is then released from the DNA by heat denaturation before the first PCR cycle. Through this mechanism, PCR would be enhanced by reducing off-target extension at room temperature.

Evaluating the Efficiency of REPLI-g® Single Cell Kit for Trace DNA Amplification.

Objective To evaluate the efficiency of REPLI-g® Single Cell Kit for sample DNA amplification, and explore its application value in forensic trace DNA amplification. Methods Three DNA extraction kits were selected to extract DNA from peripheral blood of 10 unrelated individuals. The DNA yield and purity of the three DNA extraction kits were compared. According to the results of comparison, one DNA sample was selected to concentrate and dilute, then used as the initial sample of whole genome amplification （WGA）. REPLI-g® Single Cell Kit was used to amplify the initial sample at the whole genome level. The amplification yield and amplification times were calculated, and the distribution of DNA fragments was detected by agarose gel electrophoresis. Goldeneye® DNA ID System 20A Kit was used to perform the STR typing of the initial sample and DNA samples amplified at the whole genome level to evaluate the performance of REPLI-g® Single Cell Kit in trace DNA amplication in terms of purity and yield as well as the success rate of STR typing. Results After comparison, one DNA sample was selected from QIAsymphony® DNA Investigator® Kit extracts to concentrate and dilute as the initial sample of WGA. After amplifying the whole genome of a series of initial samples by REPLI-g® Single Cell Kit, the lowest average of amplification yield reached 8.77×103 ng, while the average of the corresponding amplification times reached 1.40×106. DNA fragments were large and concentrated. The STR typing success rate of WGA samples became lower with the decrease of initial samples used, but when the amount of samples was lower than 0.5 ng, the STR typing success rate of samples after DNA WGA was higher than that of samples without DNA WGA. Conclusion REPLI-g® Single Cell Kit can increase the yield of template DNA. Especially for trace DNA, the STR typing success rate can be improved to a certain extent.

Metabarcoding for stomach-content analyses of Pygmy devil ray (Mobula kuhlii cf. eregoodootenkee): Comparing tissue and ethanol preservative-derived DNA.

The application of high‐throughput sequencing to retrieve multi‐taxon DNA from different substrates such as water, soil, and stomach contents has enabled species identification without prior knowledge of taxon compositions. Here we used three minibarcodes designed to target mitochondrial COI in plankton, 16S in fish, and 16S in crustaceans, to compare ethanol‐ and tissue‐derived DNA extraction methodologies for metabarcoding. The stomach contents of pygmy devilrays (Mobula kuhlii cf. eregoodootenkee) were used to test whether ethanol‐derived DNA would provide a suitable substrate for metabarcoding. The DNA barcoding assays indicated that tissue‐derived operational taxonomic units (OTUs) were greater compared to those from extractions performed directly on the ethanol preservative. Tissue‐derived DNA extraction is therefore recommended for broader taxonomic coverage. Metabarcoding applications should consider including the following: (i) multiple barcodes, both taxon specific (e.g., 12S or 16S) and more universal (e.g., COI or 18S) to overcome bias and taxon misidentification and (ii) PCR inhibitor removal steps that will likely enhance amplification yields. However, where tissue is limited or no longer available, but the ethanol‐preservative medium is still available, metabarcoding directly from ethanol does recover the majority of common OTUs, suggesting the ethanol‐retrieval method could be applicable for dietary studies. Metabarcoding directly from preservative ethanol may also be useful where tissue samples are limited or highly valued; bulk samples are collected, such as for rapid species inventories; or mixed‐voucher sampling is conducted (e.g., for plankton, insects, and crustaceans).

Enzymatic Preparation of Modified DNA: Study of the Kinetics by Real-Time PCR

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).

A multi-channel polymerase chain reaction lab-on-a-chip and its application in spaceflight experiment for the study of gene mutation

Astronauts face serious health threats during spaceflight. The two major factors that may lead to an astronauts' physiological dysfunction are space radiation and microgravity. Heavy ion radiation, one of the important components of charged particles in orbit, can cause deoxyribonucleic acid (DNA) damage and gene mutations. Microgravity can also affect a series of cell physiology functions, including cytoskeleton remodeling, DNA modification, interactions between molecules, etc. Several ionizing radiation experiments performed in our previous study suggested the variation in the mutation across different selected immune genes. Here, we performed a further experiment (IGM-BIT-1) on board the International Space Station (ISS) to explore the molecule evolution rules of the selected DNA. To conduct the on-orbit amplification of the DNA fragments from the antibody encoding genes in the ISS, a self-developed portable and programmable PCR device was designed and produced. We developed a novel PCR chip that consists of a multi-channel optical adhesive reaction chamber and a miniature thermal cycler. The reaction chamber was cost effective and disposable. The thermal cycler was used to achieve both rapid heating and cooling. As the DNA amplification yield of IGM-BIT-1 PCR device was much similar or even higher than the commercial devices, the IGM-BIT-1 payload has been proven to be suitable for space life science research.