Biomolecular Detection and Quantification最新文献

The kinetic requirements of quantitative PCR were experimentally dissected into the stages of DNA denaturation, primer annealing, and polymerase extension. The temperature/time conditions for 2 stages were kept optimal, while the other was limited until the amplification efficiency decreased as measured by an increase in quantification cycle (Cq). Extension was studied in a commercial capillary LightCycler®. Using a rapid deletion mutant of Taq (KlenTaq^™), about 1 s was required for every 70 bp of product length. To study annealing and denaturation times of <1 s, a custom “extreme” PCR instrument with 3 temperatures was used along with increased primer and polymerase concentrations. Actual sample temperatures and times were measured rather than programmed or predicted. For denaturation, 200–500 ms above the denaturation threshold was necessary for maximal efficiency. For annealing, 300-1000 ms below the annealing threshold was required. Temperature thresholds were set at 98% primer annealing or PCR product denaturation as determined experimentally by melting curves. Progressing from rapid cycle PCR to extreme PCR decreased cycling times by 10–60 fold. If temperatures are controlled accurately and flexibility in reagents is allowed, PCR of short products can be performed in less than 15 s. We also put PCR in context to other emerging methods and consider its relevance to the evolution of molecular diagnostics.

Non-coding RNAs were established in the last decade as a new valuable biomarker class for human diseases. Specifically, circular RNAs (circRNAs) were only recently discovered as a new large group of non-coding RNAs that, due to their circular configuration, are metabolically more stable compared to their linear counterparts and therefore highly suitable for biomarker use. Based on high-throughput sequencing, the catalogs of endogenous circRNAs with disease relevance and correlation continue to grow steadily. As a consequence, circRNAs emerged as novel and attractive biomarkers, indicated by numerous recent publications. Here we would like to stress the need of essential quality criteria for validation and characterization of circular RNAs. In addition to high-throughput sequencing, classical biochemical methods are essential and should be applied for the characterization of this special class of RNAs, in particular to convincingly confirm their circularity.

Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand, cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on patent sequence information, event-specific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed. Newly developed plasmids were used as reference material for assay optimization and in-house validation. Plasmid standards were quantified using digital droplet PCR and LOD95%, PCR efficiency, robustness and specificity of the assays were determined using real-time PCR. A LOD95% of 10 copies per PCR reaction was observed and PCR efficiencies of 95–97 % were achieved. Different real-time PCR instruments and PCR conditions were applied to test for robustness of the assays using DNA at a concentration of 30 copies per μL for each gm alfalfa event. All replicates were positive independent of the instrument or the PCR condition. DNA from certified reference material of different genetically modified crops as well as reference materials of the three events was used to experimentally test for specificity. No unspecific amplification signal was observed for any of the assays. Validation results were in line with the “Minimum Performance Requirements for Analytical Methods of GMO Testing” of the European Network of GMO Laboratories. Furthermore, an inter-laboratory comparison study was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters.

Despite the wide-spread use of the polymerase chain reaction (PCR) in various life-science applications, the causes of arrested amplicon generation in late cycles have not been confidently identified. This so-called plateau phase has been attributed to depletion or thermal break-down of primers or nucleotides, thermal inactivation of the DNA polymerase, and product accumulation resulting in competition between primer annealing and product re-hybridization as well as blocking of DNA polymerase by double-stranded amplicons. In the current study, we experimentally investigate the proposed limiting factors of PCR product formation. By applying robust and validated qPCR assays, we elucidate the impact of adding non-target and target amplicons to the reactions, mimicking the high amount of products in late PCR cycles. Further, the impact of increased primer concentrations and thermal stability of reagents are explored. Our results show that high amounts of non-target amplicons inhibit amplification by binding to the DNA polymerase, but that this effect is counteracted by addition of more DNA polymerase or prolonged annealing/extension times. Adding high amounts of target amplicons that also act as templates in the reaction is far less inhibitory to amplification, although a decrease in amplification rate is seen. When primer concentrations are increased, both amplification rates and end-product yields are elevated. Taken together, our results suggest that the main cause of PCR plateau formation is primer depletion and not product accumulation or degradation of reagents. We stress that a PCR plateau caused by primer depletion is assay-dependent, i.e. dependent on the primer design and primer characteristics such as the probability of primer-dimer formation. Our findings contribute to an improved understanding of the major parameters controlling the PCR dynamics at later cycles and the limitations of continued product formation, which in the end can facilitate PCR optimization.

Over the last few decades, there has been a decline in Northern bobwhite quail (Colinus virginianus) throughout their native range. While there are various factors that may be influencing this decline, it is suggested that parasites should be taken into consideration as a potential contributor in the Rolling Plains Ecoregion. High prevalence of the eyeworm (Oxyspirura petrowi) and caecal worm (Aulonocephalus pennula) in bobwhite of this region, coupled with a continuous decline, creates a need to assess infection through alternative methods for regional surveillance. Previous studies have developed a qPCR method and mobile research laboratory as an option for nonlethal procedures. However, there is still a need for standardization of these techniques. Therefore, this study builds on previous protocols to develop an application that considers factors that may influence qPCR results. In this study, cloacal swabs are collected from bobwhite in three locations throughout the Rolling Plains and scaled based on amount of feces present on the swab. This data is compared to qPCR standards as a limit of quantification for both eyeworm and caecal worm to define parasitic infection levels. Binary logistic regressions confirm that the probability of detection increases for both eyeworm (Odds Ratio: 2.3738; 95% Confidence Interval: [1.7804, 3.1649]) and caecal worm (Odds Ratio: 2.8516; 95% Confidence Interval: [2.2235, 3.6570]) as swab score increases. Infection levels for eyeworm and caecal worm are based on the generated cycle threshold value averages of qPCR standards. Based on the results of this study, this method can be applied in the mobile research laboratory to quantitatively assess regional parasitic infection in bobwhite throughout the Rolling Plains.

Objectives

Detection of genomic alterations in diseases can be achieved with current molecular technologies. However, the molecules extracted from formalin-fixed, paraffin-embedded (FFPE) bio-samples are often limited possibly due to DNA fragmentation and crosslinking caused by the sample fixation and processing. The study objective was to design a droplet digital PCR (ddPCR) assay to assess the quality and quantity of DNA derived from various DNA extraction conditions on FFPE samples.

Methods

We used 10 μm-thick sections from 5 FFPE oral tumoral blocks, each consisting of 10–15 sections. The protocol variables tested included: 1) tissue staining; 2) duration and 3) temperature of post-digestion heat treatment; and 4) DNA extraction method. DNA quantity was assessed using the NanoDrop 2000 (Thermo Fisher Scientific, USA), the Qubit fluorometer (Thermo Fisher Scientific, USA), and a ddPCR-based assay. DNA quality was assessed using a ddPCR assay for the degree of fragmentation and the effectiveness of removing crosslinks with varying guanine-cytosine (GC)-content.

Results

Deparaffinization with xylene helped to increase the DNA yield. Tissue staining (methyl green staining, pH 6) prior to microdissection, comparing to no staining, caused additional DNA fragmentation. Compared to column-based method, DNA extracted with phenol chloroform and ethanol precipitation increased the degree of fragmentation and lowered the yield of amplifiable DNA. The cross-linking derived from GC-contents may not be the only factor impacting on the DNA quality.

Conclusions

Samples undergoing different pre-treatment conditions prior to extraction can impact the yield of amplifiable DNA. Our ddPCR assay can be used to assess for both DNA quantity and quality.

Amplification curves from quantitative Real-Time PCR experiments typically exhibit a sigmoidal shape. They can roughly be divided into a ground or baseline phase, an exponential amplification phase, a linear phase and finally a plateau phase, where in the latter, the PCR product concentration no longer increases. Nevertheless, in some cases the plateau phase displays a negative trend, e.g. in hydrolysis probe assays. This cycle-to-cycle fluorescence decrease is commonly referred to in the literature as the hook effect. Other detection chemistries also exhibit this negative trend, however the underlying molecular mechanisms are different.

In this study we present two approaches to automatically detect hook effect-like curvatures based on linear (hookreg) and nonlinear regression (hookregNL). As the hook effect is typical for qPCR data, both algorithms can be employed for the automated identification of regular structured qPCR curves. Therefore, our algorithms streamline quality control, but can also be used for assay optimization or machine learning.

The quantitative real-time polymerase chain reaction (qPCR) is one of the most commonly molecular methods used today. It is central to numerous assays that have since been developed and described around its optimization. The Listeria monocytogenes prfA qPCR assay has been studied in great detail and due to its comprehensive knowledge, excellent performance (sensitivity of one single copy), and internal amplification control, it represents a suitable test platform for qPCR examinations. In this study, we compared ten different polymerases (or ready-to-use mastermixes) as possible (economic) alternatives to our gold standard Platinum Taq polymerase. We sought to determine the reproducibility of these assays under modified conditions, which are realistic because published assays are frequently used with substituted polymerases. Surprisingly, there was no amplification at all with some of the tested polymerases, even although the internal amplification control worked well. Since adaptation of the thermal profile and of MgCl₂ concentration could restore amplification, simple replacement of the polymerase can destroy a well-established assay leading up to >10⁶-fold less analytical sensitivity. Further, validation using Poisson and PCR-Stop analyses revealed limits to some assay-polymerase combinations and emphasize the importance of validation.