DNA Primers Research Articles

Characterization of genetic diversity in Curcuma longa L. cultivars using Start Codon Targeted (SCoT) markers

Curcuma longa L. (Family- Zingiberaceae), commonly called turmeric, is one of the most important commercial spices. Being a center of turmeric's diversity, India boasts of more than fifty cultivars with variable agronomic characters and curcumin content. However, the quest for calibrating their genetic variability remains debatable as popular molecular marker-based studies have yielded inconsistent results. A promising PCR-based approach, known as the Start Codon Targeted (SCoT)-PCR system has been established to be more efficient than random DNA marker systems because this technique can generate more information on genetic polymorphism patterns. Hence, the present study proposes to use Start Codon Targeted (SCoT) markers to assess inter-cultivar genetic diversity of turmeric. We report an orthogonal L16 (45) design-based approach to investigate the influence of five crucial factors (dNTPs, Taq DNA polymerase, primer, template DNA and annealing temperature of primer) on the efficiency of the polymerase chain reaction (PCR) for development of an optimized Start Codon Targeted (SCoT) amplification protocol using twenty-seven different cultivars of C. longa. The optimized PCR protocol yields best results with a 20 uL reaction mixture, containing 10×PCR Buffer (with 15 mM MgCl2), 2.5 mM dNTPs, 10 mM SCoT primer, Taq polymerase (2 U), template DNA (50 ng), which was verified by using twenty-five SCoT primers. This standardized rapid protocol of SCoT-PCR reported here was employed to examine genetic diversity among twenty-seven Indian cultivars of turmeric, revealing high range of polymorphism ranging from 81.25 % to 100 %. Unweighted pair group method of arithmetic averages (UPGMA) cluster analysis of the SCoT marker data segregated the twenty-seven cultivars into two main clusters. The first cluster comprised of nine cultivars (36 % of the cultivars) which was further divided into three sub-clusters and the second one consisted of eighteen cultivars (64 %) which further divided into four sub-clusters. From the results of this study, it can be inferred that the employment of SCoT markers can be an effective tool for analysing genetic variability, phylogenetic relationships and develop a rich genetic resource management system for C. longa cultivars.

The oxidation of the [4Fe-4S] Cluster of DNA Primase Alters the Binding Interactions with DNA and RNA Primers

DNA primase is an iron sulfur enzyme in DNA replication, responsible for synthesizing short RNA primers that serve as starting points for DNA synthesis. The role of the [4Fe-4S] cluster is not well determined. Here, we calculate the redox potential (Em) of the [4Fe-4S] with and without DNA/RNA using continuum electrostatics. In addition, we identify the structural changes coupled to the oxidation/reduction. Our calculations show that the DNA/RNA primer lowers the Em by 110 mV and 50 mV for the [4Fe-4S]+ and [4Fe-4S]2+ states respectively. The oxidation of the cluster is coupled to structural changes that significantly reduces the binding energies between the DNA and the nearby residues. The negative charges accumulated by the DNA and the RNA primers induce the oxidation of the [4Fe-4S] cluster. This in turn stimulates structural changes on the DNA-protein interface that significantly reduces the binding energies.

Polymerase-Based Signal Delay for Temporally Regulating DNA Involved Reactions, Programming Dynamic Molecular Systems, and Biomimetic Sensing.

Complex temporal molecular signals play a pivotal role in the intricate biological pathways of living organisms, and cells exhibit the ability to transmit and receive information by intricately managing the temporal dynamics of their signaling molecules. Although biomimetic molecular networks are successfully engineered outside of cells, the capacity to precisely manipulate temporal behaviors remains limited. In this study, the catalysis activity of isothermal DNA polymerase (DNAP) through combined use of molecular dynamics simulation analysis and fluorescence assays is first characterized. DNAP-driven delay in signal strand release ranged from 100 to 102min, which is achieved through new strategies including the introduction of primer overhangs, utilization of inhibitory reagents, and alteration of DNA template lengths. The results provide a deeper insight into the underlying mechanisms of temporal control DNAP-mediated primer extension and DNA strand displacement reactions. Then, the regulated DNAP catalysis reactions are applied in temporal modulation of downstream DNA-involved reactions, the establishment of dynamic molecular signals, and the generation of barcodes for multiplexed detection of target genes. The utility of DNAP-based signal delay as a dynamic DNA nanotechnology extends beyond theoretical concepts and achieves practical applications in the fields of cell-free synthetic biology and bionic sensing.

Aberrant methylation of placental development genes in chorionic villi of spontaneous abortions with trisomy 16.

In humans, aneuploidy is incompatible with the birth of healthy children and mainly leads to the death of embryos in the early stages of development in the first trimester of pregnancy. Trisomy 16 is the most common aneuploidy among spontaneous abortions of the first trimester of pregnancy. However, the mechanisms leading to the death of embryos with trisomy 16 remain insufficiently investigated. One of these potential mechanisms is abnormal placental development, including aberrant remodeling of spiral arteries. Spiral artery remodeling involves the migration of trophoblast cells into the maternal spiral arteries, replacing their endothelium and remodeling to ensure a stable embryonic nutrition and oxygen supply. This is a complex process which depends on many factors from both the embryo and the mother. We analyzed the methylation level of seven genes (ADORA2B, NPR3, PRDM1, PSG2, PHTLH, SV2C, and TICAM2) involved in placental development in the chorionic villi of spontaneous abortions with trisomy 16 (n = 14), compared with spontaneous abortions with a normal karyotype (n = 31) and the control group of induced abortions (n = 10). To obtain sequencing libraries, targeted amplification of individual gene regions using designed oligonucleotide primers for bisulfite-converted DNA was used. The analysis was carried out using targeted bisulfite massive parallel sequencing. In the group of spontaneous abortions with trisomy 16, the level of methylation of the PRDM1 and PSG2 genes was significantly increased compared to induced abortions (p = 0.0004 and p = 0.0015, respectively). In the group of spontaneous abortions, there was no increase in the level of methylation of the PRDM1 and PSG2 genes, but the level of methylation of the ADORA2B gene was significantly increased compared to the induced abortions (p = 0.032). The results obtained indicate the potential mechanisms of the pathogenetic effect of trisomy 16 on the placental development with the participation of the studied genes.

331 Validation of a Novel CSF-Based Biomarker of Mitochondrial Function

OBJECTIVES/GOALS: Determine the exosome mitochondrial DNA (mtDNA) copy number in cerebrospinal fluid (CSF) as a measure of neuronal mitochondrial integrity in patients with subarachnoid hemorrhage (SAH). Determine the patterns of beta amyloid and tau protein biology in CSF of SAH patients and correlate those measures with the clinical status of the SAH patients. METHODS/STUDY POPULATION: The CSF is collected from SAH patients undergoing ventriculostomy-based continuous CSF drainage. Adults from all ethnicities and sex are included in this study. The exosomes are isolated from CSF samples using a precipitation method and particle count and size are measured using NanoSight. The DNA is extracted using an exosomal DNA isolation kit (XCF kit). The CSF mtDNA copy number is measured using digital drop PCR with mitochondrial DNA primers. The levels of beta-amyloid (a-beta-40 and -42) and tau protein in CSF are measured using a sensitive ELISA-based assay. A quantitative evaluation of mitochondrial DNA copy number, clinical status of the SAH patients and beta amyloid, and tau protein levels will be conducted and reported. RESULTS/ANTICIPATED RESULTS: Preliminary results of four CSF samples showed similar patterns in CSF exosome particle number, particle size and exosomal mtDNA copy number in relation to samples from the admission day. Particle number decreased with time while particle size increased. More patient samples will be analyzed to confirm the patterns. We anticipate that mtDNA copy number will correlate with brain beta-amyloid and tau protein levels. Moreover, we anticipate that the clinical status of the SAH patients will associate with the mtDNA copy number. We specifically predict that higher mtDNA copy number levels will correlate with better clinical outcomes. DISCUSSION/SIGNIFICANCE: Mitochondrial function is critical to brain health, but we lack effective ways to monitor this parameter. Here we focus on a CSF based biomarker, exosome-derived mtDNA, which is intended to reflect the integrity of brain mitochondria. As bioenergetic metabolism influences beta amyloid and tau biology, predicting those levels are important.

Real-Time Monitoring of a Nucleic Acid Amplification Reaction Using a Mass Sensor Based on a Quartz-Crystal Microbalance.

Nucleic acid amplification reactions such as polymerase chain reaction (PCR), which uses a DNA polymerase to amplify individual double-stranded DNA fragments, are a useful technique for visualizing the presence of specific genomes. Although the fluorescent labeling method is mainly used with DNA amplification, other detection methods should be considered for further improvements, such as miniaturization and cost reduction, of reaction-monitoring devices. In this study, the quartz-crystal microbalance (QCM) method, which can measure nanogram-order masses, was applied for the real-time detection of DNA fragments in a solution with nucleic acids. This was combined with an isothermal nucleic acid amplification reaction based on the recombinase polymerase amplification (RPA) method, which allowed DNA amplification at a constant temperature. When the DNA amplification reaction was initiated on a QCM sensor plate with an immobilized primer DNA strand, a significant increase in mass was observed compared to when the primer DNA was not immobilized. QCM was shown to be sufficiently sensitive for the in situ detection of amplified DNA fragments. Combining a portable QCM device and RPA offers a sensitive point-of-care method for detecting nucleic acids.

Detection of terminal deoxynucleotidyl transferase activity based on self-mediated nucleic acid elongation and elemental labeling inductively coupled plasma-mass spectrometry

Terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase, is recognized as a promising biomarker for acute leukemia. Herein, taking the advantage of the self-mediated strand elongation property of TdT, a simple and sensitive method for TdT activity assay was developed based on gold nanoparticles (AuNPs) labeling inductively coupled plasma mass spectrometry (ICP-MS). In the presence of TdT, the primer DNA on magnetic beads is elongated with an adenine-rich single stranded long chain that can label poly-thymine modified AuNPs. After acid elution, the labeled AuNPs were detected by ICP-MS, and the signal intensity of 197Au reflected the TdT activity. Under the optimal conditions, the limit of detection for TdT activity is down to 0.054 U mL−1, along with good selectivity and strong tolerance to other interfering proteins. Furthermore, it achieves a straightforward and accurate detection of TdT activity in acute lymphoblastic leukemia cells without sample pre-processing and tool enzyme addition. Therefore, the proposed method shows great promise as a valuable tool for TdT-related biological research and leukemia therapeutics.

Genetic diversity of maize resources revealed by different molecular markers

AbstractMaize (Zea mays L.) is the third most important cereal crop in the world because of its nutritional value and industrial benefits. Molecular markers are used mainly by the breeders to study the genetic variability of genotypes and its application in the breeding process. Two types of molecular markers, 10 random amplified polymorphic DNA (RAPD) primers and 10 start codon target (SCoT) primers, were assayed to determine the genetic diversity of 25 Slovak maize lines and 25 maize cultivars. A high level of polymorphism was found with both RAPD and SCoT markers, which was confirmed by high average polymorphism information content (PIC) values using both techniques. The efficiency of individual marker techniques in the detection of genotype diversity can be compared by calculating the marker index (MI), detecting diversity index (DDI), discriminating power, resolving power (RP) and other indices. A higher MI (11.788), DDI (2.358) and RP (53.08) value was achieved by the SCoT technique compared to the RAPD method. Three joint dendrograms and PCoA plots constructed based on RAPD, SCoT and both methods combined confirmed the unambiguous separation of maize lines and cultivars from each other. The results obtained from the RAPD and SCoT analysis can be used for the selection of potentially suitable biological sources for further marker-assisted breeding.

Impact of ChAdOx1 or DNA Prime Vaccination on Magnitude, Breadth, and Focus of MVA-Boosted Immunogen-Specific T Cell Responses.

The efficacy of anti-viral T-cell vaccines may greatly depend on their ability to generate high-magnitude responses targeting a broad range of different epitopes. Recently, we created the HIV T-cell immunogen HTI, designed to generate T-cell responses to protein fragments more frequently targeted by HIV controllers. In the present study, we aim to maximize the breadth and magnitude of the T-cell responses generated by HTI by combining different vaccine vectors expressing HTI. We evaluated the ability to induce strong and broad T-cell responses to the HTI immunogen through prime vaccination with DNA plasmid (D) or Chimpanzee Adenovirus Ox1 (ChAdOx1; C) vectors, followed by a Modified Virus Ankara (MVA; M) vaccine boost (DDD, DDDM, C, and CM). HTI-specific T-cell responses after vaccination were measured by IFN-γ-ELISpot assays in two inbred mice strains (C57BL/6 and BALB/c). CM was the schedule triggering the highest magnitude of the response in both mice strains. However, this effect was not reflected in an increase in the breadth of the response but rather in an increase in the magnitude of the response to specific immunodominant epitopes. Immunodominance profiles in the two mouse strains were different, with a clear dominance of T-cell responses to a Pol-derived peptide pool after CM vaccination in C57BL/6. Responses to CM vaccination were also maintained at higher magnitudes over time (13 weeks) compared to other vaccination regimens. Thus, while a ChAdOx1 prime combined with MVA booster vaccination generated stronger and more sustained T-cell responses compared to three DNA vaccinations, the ChAdOx1 primed responses were more narrowly targeted. In conclusion, our findings suggest that the choice of vaccine vectors and prime-boost regimens plays a crucial role in determining the strength, duration, breadth, and focus of T-cell responses, providing further guidance for selecting vaccination strategies.

Field-effect transistor biosensor with signal amplification by ternary initiation complexes for detection of wide-range RNA concentration

Electrical detection of RNAs using transistor-based biosensors has attracted attention as a strategy for medical diagnosis and environmental monitoring. Herein, we demonstrated a proof-of-concept for specific, sensitive, and label-free RNA detection using a field-effect transistor (FET) biosensor with signal amplification by ternary initiation complexes (SATIC), which is an isothermal one-step nucleic acid amplification initiated by the combination of target RNA, circular DNA template and DNA primer. The SATIC system-applied FET biosensor specifically and quantitatively detected the target RNA with a single-nucleotide difference via the negative charges derived from the amplification products formed by a nucleic acid amplification reaction with φ29 DNA polymerase on the gate surface. In particular, the control of the amplification time allowed the detection of target RNA molecules over a wide concentration range, resulting in a detection limit of up to 6 copies/μL. Therefore, a transistor-based bioassay using the SATIC system could be useful for simple and sensitive nucleic acid analysis.

MOLECULAR DETECTION OF WATER PROTOZOAN PARASITES FROM SELECTED RIVERS IN RIVERS STATE, NIGERIA

Drinking water or potable water also known as improved drinking water is said to be any water that is free from physical, chemical, biological or radiological form of contamination. Water samples were collected from 5 rivers in Omuchi Aluu, Choba, Chokocho, Oyigbo, and Aleto communities, in Rivers State, Nigeria, used for activities such as washing, bathing, fishing, and dredging by community indigenes. Water samples were collected from various sampling stations and, transported to the laboratory, African Biosciences, Ibadan for analysis to characterize and identify the protozoan parasites using molecular techniques. This study aimed at characterizing the genotype of protozoan parasites of water from selected rivers in River State, Nigeria, using molecular methods to check for portability and purity of water. DNA Primers of five protozoan parasites, Giardia species, Entamoeba species, Cryptosporidium species, Cyclospora species, and Toxoplasma species were used to analyze and detect the presence of the genes of these protozoans. The study showed the presence of Giardia duodenalis and Cryptosporidium parvuum, and the absence of Entamoeba histolytica, Cyclospora cayetanensis, and Toxoplasma gondii from the water samples collected from Omuchi Aluu, Choba, Chokocho, Oyigbo, and Aleto rivers. It was concluded that water samples collected from rivers contaminated with parasites, is unsuitable for drinking, hence community locals should be sensitized to embark on good water sanitary and hygiene (WASH) practices, for a healthy life and sustainable environment.

DNA digestion and formation of DNA-network structures with Holliday junction-resolving enzyme Hjc_15-6 in conjunction with polymerase reactions

The recently identified novel Holliday junction-resolving enzyme, termed Hjc_15-6, activity investigation results imply DNA cleavage by Hjc_15-6 in a manner that potentially enhances the molecular self-assembly that may be exploited for creating DNA-networks and nanostructures. The study also demonstrates Pwo DNA polymerase acting in combination with Hjc_15-6 capability to produce large amounts of DNA that transforms into large DNA-network structures even without DNA template and primers. Furthermore, it is demonstrated that Hjc_15-6 prefers Holliday junction oligonucleotides as compared to Y-shaped oligonucleotides as well as efficiently cleaves typical branched products from isothermal DNA amplification of both linear and circular DNA templates amplified by phi29-like DNA polymerase. The assembly of large DNA network structures was observed in real time, by transmission electron microscopy, on negative stained grids that were freshly prepared, and also on the same grids after incubation for 4 days under constant cooling. Hence, Hjc_15-6 is a promising molecular tool for efficient production of various DNA origamis that may be implemented for a wide range of applications such as within medical biomaterials, catalytic materials, molecular devices and biosensors.

Transient states during the annealing of mismatched and bulged oligonucleotides.

Oligonucleotide hybridization is crucial in various biological, prebiotic and nanotechnological processes, including gene regulation, non-enzymatic primer extension and DNA nanodevice assembly. Although extensive research has focused on the thermodynamics and kinetics of nucleic acid hybridization, the behavior of complex mixtures and the outcome of competition for target binding remain less well understood. In this study, we investigate the impact of mismatches and bulges in a 12bp DNA or RNA duplex on its association (kon) and dissociation (koff) kinetics. We find that such defects have relatively small effects on the association kinetics, while the dissociation kinetics vary in a position-dependent manner by up to 6 orders of magnitude. Building upon this observation, we explored a competition scenario involving multiple oligonucleotides, and observed a transient low specificity of probe hybridization to fully versus partially complementary targets in solution. We characterize these long-lived metastable states and their evolution toward equilibrium, and show that sufficiently long-lived mis-paired duplexes can serve as substrates for prebiotically relevant chemical copying reactions. Our results suggest that transient low accuracy states may spontaneously emerge within all complex nucleic acid systems comprising a large enough number of competing strands, with potential repercussions for gene regulation in the realm of modern biology and the prebiotic preservation of genetic information.

Exploring the Impact of Primer-Template Mismatches on PCR Performance of DNA Polymerases Varying in Proofreading Activity.

Polymerase chain reaction (PCR) is a widely used technique in gene expression analysis, diagnostics, and various molecular biology applications. However, the accuracy and sensitivity of PCR can be compromised by primer-template mismatches, potentially leading to erroneous results. In this study, we strategically designed 111 primer-template combinations with varying numbers, types, and locations of mismatches to meticulously assess their impact on qPCR performance while two distinctly different types of DNA polymerases were used. Notably, when a single-nucleotide mismatch occurred at the 3' end of the primer, we observed significant decreases in the analytical sensitivity (0-4%) with Invitrogen™ Platinum™ Taq DNA Polymerase High Fidelity, while the analytical sensitivity remained unchanged with Takara Ex Taq Hot Start Version DNA Polymerase. Leveraging these findings, we designed a highly specific PCR to amplify Babesia while effectively avoiding the genetically close Theileria. Through elucidating the critical interplay between types of DNA polymerases and primer-template mismatches, this research provides valuable insights for improving PCR accuracy and performance. These findings have important implications for researchers aiming to achieve robust qPCR results in various molecular biology applications.

Effect of chloride salt types on corrosion resistance of reinforcing steel in cement mortar mixed with DNA primer inhibitor

It is important to study the corrosion resistance of reinforcing steel in various chloride salt environment. In this study, the corrosion performance of reinforcing steel inside mortar mixed with a new deoxyribonucleic acid (DNA) primer inhibitor in various chloride salt solutions (NaCl, KCl, CaCl2, MgCl2) was investigated. The results indicate that the chloride salt types have a significant effect on corrosion resistance of reinforcing steel in cement mortar mixed with DNA primer inhibitor, and the rule is NaCl ≈ KCl > CaCl2 >MgCl2. The new DNA primer inhibitor can maintain the good corrosion inhibition performance in cement mortar. NaCl and KCl can inhibit DNA primer inhibitor condensation and dissolution of calcium hydroxide, which can promote the formation of a better protective film compared with CaCl2 and MgCl2. MgCl2 and CaCl2 can lead to relatively high porosity and accelerate the diffusion of chloride ions to the steel surface than NaCl and KCl.

Ultra-low volume intradermal administration of radiation-attenuated sporozoites with the glycolipid adjuvant 7DW8-5 completely protects mice against malaria

Radiation-attenuated sporozoite (RAS) vaccines can completely prevent blood stage Plasmodium infection by inducing liver-resident memory CD8+ T cells to target parasites in the liver. Such T cells can be induced by ‘Prime-and-trap’ vaccination, which here combines DNA priming against the P. yoelii circumsporozoite protein (CSP) with a subsequent intravenous (IV) dose of liver-homing RAS to “trap” the activated and expanding T cells in the liver. Prime-and-trap confers durable protection in mice, and efforts are underway to translate this vaccine strategy to the clinic. However, it is unclear whether the RAS trapping dose must be strictly administered by the IV route. Here we show that intradermal (ID) RAS administration can be as effective as IV administration if RAS are co-administrated with the glycolipid adjuvant 7DW8-5 in an ultra-low inoculation volume. In mice, the co-administration of RAS and 7DW8-5 in ultra-low ID volumes (2.5 µL) was completely protective and dose sparing compared to standard volumes (10–50 µL) and induced protective levels of CSP-specific CD8+ T cells in the liver. Our finding that adjuvants and ultra-low volumes are required for ID RAS efficacy may explain why prior reports about higher volumes of unadjuvanted ID RAS proved less effective than IV RAS. The ID route may offer significant translational advantages over the IV route and could improve sporozoite vaccine development.

OPTIMISATION OF PRIMER CONCENTRATION AND ANNEALING TEMPERATURE IN PCR TEST METHOD FOR AFRICAN SWINE FEVER VIRUS DETECTION

African Swine Fever disease causes high fatality number in domestic pigs. The most sensitive and specific laboratory diagnotic is PCR. The successful of PCR is determined by DNA quality, Annealing temperature, MGCl2 concentration, polymerase enzymes, primer concentration, and PCR cycles. The Annealing process requires in the right temperature. The difference annealing temperature causes amplification process failure. The specificity and sensitivity of PCR products are determined by the optimal annealing temperature. PCR optimization, variations of time, temperature, and primer concentration can be adjusted. This study was conducted by PCR testing to detect DNA ASF virus using primer concentration and the most optimum Annealing temperature. Identification of primer concentrations and annealing temperature begins to make PCR components volume 10 µl consist of 5µL PCR Master Mix, a pair of ASF primer 0.5 µl, 1µL DNA templates, and 3µL ddH2O. The PCR component is processed in a thermocycler with a temperature condition of 95°C pre-denaturation for 5 minutes, and denaturation at 94°C for 45 seconds. The gradient annealing temperature starts at 50°C, 52°C, 55°C, 59°Cm and 65°C for 1 minute. Temperature 72 ° C for synthesis 1 minute. repetition of the cycle is 34 times. Final synthesis for 72°C for 5 minutes, the storage temperature is 22°C. The result of optimization at the primer concentration of 10 µm obtained a cleared band according to the target compared to the primary concentration of 5 µm. The best annealing temperature in this research is 55 °C. If the Annealing temperature is given lower, causing primer attachment to the DNA template is not specific so non-specific PCR products are formed many bands. Conversely, if the annealing temperature is higher, causing primary attachment to the template DNA will be released so that the band that appears is very thin PCR product is not formed does not appear.

DNA metabarcoding diet analysis in a generalist omnivore: feeding trials reveal the efficacy of extraction kits and a multi-locus approach for identifying diverse diets.

Metabarcoding-based diet analysis is a valuable tool for understanding the feeding behavior of a wide range of species. However, many studies using these methods for wild animals assume accuracy and precision without experimental evaluation with known positive control food items. Here, we conducted a feeding trial experiment with a positive control community in pasture-raised chickens and assessed the efficacy of several commonly used DNA extraction kits and primer sets. We hand-fed 22 known food items, including insects and plants, to six backyard laying hens and collected their excreta for eight h. We evaluated the efficacy of three DNA extraction kits, three primer sets for plant identification (targeting rbcL, trnL, and internal transcribed spacer 2 [ITS2]), and three primer sets for arthropod identification (targeting cytochrome oxidase subunit I [COI]). The detection success rate of our positive control food items was highly variable, ranging from 2.04% to 93.88% for all kit/primer combinations and averaging 37.35% and 43.57% for the most effective kit/primer combination for plants and insects, respectively. Extraction kits using bead-based homogenization positively affected the recovery proportion of plant and insect DNA in excreta samples. The minimum time to detect known food items was 44 min post-feeding. Two COI primer sets significantly outperformed the third, and both recovery proportion and taxonomic resolution from ITS2 were significantly higher than those from rbcL and trnL. Taken together, these results display the potential variability that can be inherently present in DNA-based diet analyses and highlight the utility of experimental feeding trials in validating such approaches, particularly for omnivores with diverse diets.

Phosphoramidate Azole Oligonucleotides for Single Nucleotide Polymorphism Detection by PCR.

Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.

Could the Cytomegalovirus Viral Load Be Correlated with Cellular Rejection in Asymptomatic Heart Transplant Recipients? A Brief Report

Abstract Introduction: Cytomegalovirus (CMV) infection is mainly associated with acute or chronic graft rejection in various solid organs. This study was conducted with the aim of evaluating the plasma viral load of CMV infection in postheart transplant patients and its correlation with the grading of cellular graft rejection in endomyocardial biopsies. Materials and Methods: In a cross-sectional study, 90 samples from 65 asymptomatic heart transplant recipients scheduled for routine endomyocardial biopsy were obtained. All the recipients had a history of positive serology for CMV infection before transplantation. Evaluation of their DNA was tracked using the polymerase chain reaction technique and the use of specific virus DNA primers, and based on this, the peak virus load (the number of virus copies per milliliter) was determined. A peak viral load >10,000 copies/mL was considered positive. Graft rejection grading was determined based on the ISHLT grading system. Results: The mean (standard deviation) age of the patients (37 men) was 67 (37.3) years. The graft rejection grading was as follows: 22.2% grade 0, 38.9% Quilty effect (QE), 35.6% grade 1, and 3.3% grade 2. The mean plasma viral load level of CMV infection was 2197.4 (1256.2) μg/μL, 1970.4 (1150.3) μg/μL, and 4567.2 (1669.1) μg/μL in grades 0, QE, and 1 and/or 2, respectively (P < 0.001). The virus load was significantly higher in those with higher grades of cellular rejection. Conclusion: The relationship between the plasma level of CMV viral load and the grading of cellular graft rejection in heart transplant patients could emphasize the role of CMV virus in the pathophysiology of graft rejection. A closer surveillance for CMV infection using the viral load is also recommended in particular in those who have higher grades of graft rejection.