Template Concentration Research Articles

Convenient fabrication of conjugated polymer semiconductor nanotubes and their application in organic electronics.

Organic heterojunction is indispensable in organic electronic devices, such as organic solar cells, organic light-emitting diodes and so on. Fabrication of core–shell nanostructure provides a feasible and novel way to prepare organic heterojunction, which is beneficial for miniaturization and integration of organic electronic devices. Fabrication of nanotubes which constitute the core–shell structure in large quantity is the key for the realization of application. In this work, a simple and convenient method to prepare nanotubes using conjugated copolymer of perylene diimide and dithienothiophene (P(PDI-DTT)) was demonstrated. The relationship between preparation conditions (solvent atmosphere, solution concentration and pore diameter of templates) and morphology of nanostructure was studied systematically. P(PDI-DTT) nanotubes could be fabricated in regular shape and large quantity by preparing the solution with appropriate concentration and placing anodic aluminium oxide template with nanopore diameter of 200 nm in the solvent atmosphere. The tubular structure was confirmed by scanning electron microscopy. P(PDI-DTT) nanotubes exhibited electron mobility of 0.02 cm2 V–1 s–1 in field-effect transistors under ambient condition. Light-emitting nanostructures were successfully fabricated by incorporating tetraphenylethylene into polymer nanotubes.

High-performance nanogap electrode-based impedimetric sensor for direct DNA assays

The rapid and sensitive detection of pathogen DNA (Deoxyribonucleic acid) would be essential for diagnosis and appropriate antibiotic treatment time. Herein, we report a novel direct DNA detectable impedimetric sensor. Direct assay of the amplified target DNA (mecA gene from methicillin-resistant Staphylococcus aureus (MRSA)) was performed using the PCR (polymerase chain reaction) product without any purification. Even though there are lots of PCR reagents and excess salts in sample PCR product, the nanogap electrode-based impedimetric sensor was able to detect DNA amplification fast in 5th PCR cycle which had 260 fM mecA gene in sample originally. The 70 nm gap electrode sensor yielded over 20% signal increase at the 5th PCR cycle and the impedance change grew up to about 60% at 25th in case of sample with 260 fM mecA gene template originally. The increased concentration of target DNA template led to the rise in impedance change such as 60% up at 5th and 120% up at 25th cycle with 260 pM, respectively. It is very outstanding result as compared with the traditional PCR agarose gel. Besides, it is 7-fold superior sensitivity to the microgap electrode. Furthermore, genomic DNA sample extracted from MRSA was detected rapidly. The nanogap electrode-based impedimetric sensor could be a good candidate for a rapid, sensitive, and low-cost electrical biosensor for DNA characterization in diagnostics and disease monitoring.

A Dipolar Surface Mounted Metal-Organic Framework (SURMOF): Basis for Sensor Applications

In a cooperation within the COORNETs SPP, we aim to develop novel sensor systems based on dielectric and ferroelectric surface-mounted metal-organic frameworks (SURMOFs). To this end, we synthesize Kitagawa-type metal-organic frameworks, M2L2P (M= metal, L= linker, P= pillar) [1]. For our project, either the linker or the pillar molecules need to comprise a rotatable dipole moment perpendicular to the molecular axis. We will report on our recent results on the system [Cu2(bdc)2(dpym_btd)] (bdc2- = 1,4-benzenedicarboxylate, dpym_btd = 4,7-di(pyridine-4-yl)-2,1,3-benzothiadiazole). We have synthesized bulk MOF material using a solvothermal procedure, followed by characterization via X-ray crystallography. A layer-by-layer (LbL) approach was applied to deposit [Cu2(bdc)2(dpym_btd)] as a SURMOF on a functionalized Au(111) model surface. Several LbL parameters were optimized, such as template chemistry, deposition temperature and concentration of the precursors. Reference:[1] Kondo, T. Okubo, A. Asami, S. Noro, T. Yoshitomi, S. Kitagawa, T. Ishii, H. Matsuzaka, K. Seki, Angew. Chem. Int. Ed. 1999, 38, 140-143. Figure 1

Reproducible Enhancement of Fluorescence by Bimetal Mediated Surface Plasmon Coupled Emission for Highly Sensitive Quantitative Diagnosis of Double-Stranded DNA.

Plasmonic enhancement of fluorescence from SYBR Green I conjugated with a double-stranded DNA (dsDNA) amplicon is demonstrated on polymerase chain reaction (PCR) products. Theoretical computation leads to use of the bimetallic (Au 2 nm-Ag 50 nm) surface plasmons due to larger local fields (higher quality factors) than monometallic (Ag or Au) ones at both dye excitation and emission wavelengths simultaneously, optimizing fluorescence enhancement with surface plasmon coupled emission (SPCE). Two kinds of reverse Kretschmann configurations are used, which favor, in signal-to-noise ratio, a fluorescence assay that uses optically dense buffer such as blood plasma. The fluorescence enhancement (12.9 fold at maximum) with remarkably high reproducibility (coefficient of variation (CV) < 1%) is experimentally demonstrated. This facilitates credible quantitation of enhanced fluorescence, however unlikely to obtain by localized surface plasmons. The plasmon-induced optical gain of 46 dB due to SPCE-active dye molecules is also estimated. The fluorescence enhancement technologies with PCR enables LOD of the dsDNA template concentration of ≈400 fg µL-1 (CV < 1%), the lowest ever reported in DNA fluorescence assay to date. SPCE also reduces photobleaching significantly. These technologies can be extended for a highly reproducible and sufficiently sensitive fluorescence assay with small volumes of analytes in multiplexed diagnostics.

Solvent Free Transesterification of Glycerol Into Glycerol Carbonate Over Nanostructured CaAl Hydrotalcite Catalyst.

Drastic increase in green house gases due to fossil fuels usage urges the mankind to look for alternative fuel resources. Biodiesel is one of the alternative fuels which attracted the attention of many researchers. In recent years, bio-diesel drags much attention as an alternative clean fuel. Glycerol is an unavoidable byproduct in the transesterification process of vegetable oils into bio diesel and therefore market is flooded with glycerol. So it is high time to find ways of utilizing the abundant glycerol into value added products. Herein we report the catalytic transesterification of glycerol using dimethyl carbonate over MgAl-hydrotalcite (MgAl-HT), CaAl-hydrotalcite (CaAl-HT) and nano structured CaAl-HT catalysts. All the catalysts were characterized by XRD, FT-IR, TPD-CO2, BET, SEM and HR-TEM techniques. Among them Ca4Al-HT was found to be best in terms of conversion of glycerol (82.4%) and selectivity (95.9%) towards glycerol carbonate. The effect of CTAB template concentration in the nano synthesis of Ca4Al-HT on conversion and selectivity was studied and Ca4Al-HT synthesized with 0.4 moles of CTAB showed the best conversion of glycerol (98.7%) and the highest selectivity towards glycerol carbonate (97.9%). The recyclability test performed with the best catalyst showed that the catalyst was recyclable even after 5 cycles. Valorization of glycerol yields glycerol carbonate (GC) which is a very good polar solvent with high boiling point, building block in several organic syntheses and used in the production of surfactants, poly urethanes etc.

CuNPs decorated molecular imprinted polymer on MWCNT for the electrochemical detection of l-DOPA

A novel synthesis of copper nanoparticle (CuNPs) is carried out by the reduction of ethylene diamine Cu complexes using sodiumborohydride as reducing agent. The synthesized copper nanoparticle is grafted with molecular imprinted polymer on MWCNT (CuNPs/MWCNT-MIPs). Fabricated sorbent is used for the molecular recognition and sensing of levodopa (l-DOPA) in human urine and pharmaceutical samples. The non covalent interaction between l-DOPA and the functional groups present in the selective binding sites of the polymer composite sorbent is mainly responsible for the recognition property. The synthesis of CuNPs decorated MWCNT-MIPs and its extents are characterized by employing UV–Vis spectroscopy, Fourier-transform infrared spectroscopy, powder X-ray diffraction analysis, X-ray photoelectron spectroscopy, scanning electron microscopy and transmission electron microscopy techniques. The electrochemical investigation shows that the imprinted (CuNPs/MWCNT-MIP) material has good recognition capacity towards l-DOPA. The sensitivity is found to be directly proportional to the concentration of template with a detection limit of 7.23 × 10−9M (S/N = 3). The specificity and selectivity of the fabricated sensor give a fine discrimination between l-DOPA and structurally related compounds such as dopamine, uric acid, 3,4-Dihydroxyphenylacetic acid and homovanillic acid.

Rapid identification of Gekko gecko by loop-mediated isothermal amplification based on 12S rRNA sequence

Gekko gecko (Tokay Gecko) is a valuable traditional Chinese medicine. In this study, the loop-mediated isothermal amplification (LAMP) technique was introduced for visual rapid identification of G. gecko from adulterants. A total of sixty-five 12S rRNA sequences of fourteen species of G. gecko and its adulterants were obtained. The results showed that G. gecko could be identified from its adulterants through BLAST analysis based on 12S rRNA regions. The 12S rRNA sequences of ten batches of G. gecko were conserved. There were only two haplotypes and three variation sites in the available regions for primers design. Six specific LAMP primers were successfully designed online based on 12S rRNA sequences. The visual rapid detection of G. gecko could be achieved with the optimized conditions (64 °C for 1 h and 80 °C for 5 min). And the required minimal template concentration was 5 μg·L⁻¹ while conventional PCR with 0.5 mg·L⁻¹. Consequently, the LAMP method established from this study was rapid, specific, highly sensitive, and simple. It could be applied to detect G. gecko from its adulterants efficiently.

Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair.

The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired.

Improved Conventional PCR Assay for Detecting Tetracapsuloides bryosalmonae DNA in Fish Tissues.

Conventional PCR is an established method to detect Tetracapsuloides bryosalmonaeDNA in fish tissues and to confirm diagnosis of proliferative kidney disease (PKD) caused by T. bryosalmonae. However, the commonly used PKX5f-6r primers were designed with the intention of obtaining sequence information and are suboptimal for determining parasite DNA presence. A new PCR assay to detect T. bryosalmonae 18s rDNA, PKX18s1266f-1426r, is presented that demonstrates specificity, repeatability, and enhanced sensitivity over the PKX5f-6r assay. The limit of detection of the PKX18s1266f-1426r assay at 95% confidence was 100 template copies, and the new primers detected parasite DNA more consistently at template concentrations below 100 copies than did PKX5f-6r. The PKX18s1266f-1426r also achieved 100% detection at sample DNA concentrations one order of magnitude lower than PKX5f-6r. Out of 127 salmonid fish with unknown T. bryosalmonae infection status, PKX5f-6r detected 35 positive samples, while the new assay detected 43. The discrepancy in T. bryosalmonae detection between the two primer sets may be attributed to several differences between the assays, including oligonucleotide melting temperatures, the use of a touchdown PCR thermal cycle, and amplicon length.

Effect of template, reaction time and platinum concentration in the synthesis of PtCu/CNT catalyst for PEMFC applications

Ultra-low PtCu loading on carbon nanotubes (CNT) catalysts were synthesized by a novel galvanic displacement (GD) method under sonication conditions. The influence of the type of copper template, reaction time, and Pt concentration in the galvanic displacement bath and their correlation to electrocatalytic activity of the catalysts for oxygen reduction reaction in acidic media were studied. Among the catalysts synthesized, the best catalyst identified through electrochemical analysis was used to prepare Membrane Electrode Assembly and evaluated in a hydrogen fuel cell. The maximum power density of the PtCu/CNT and commercial catalyst (20% Pt/C) in the fuel cell were 452 mW cm−2 at 0.424 V and 358 mW cm−2 at 0.475 V, respectively, clearly showing the superior electrocatalytic capability of the PtCu/CNT catalyst synthesized in this study when compared with the commercial catalyst.

High-Nuclearity Lanthanide-Containing Clusters as Potential Molecular Magnetic Coolers.

High-nuclearity cluster-type metal complexes are a unique class of compounds, many of which have aesthetically pleasing molecular structures. Their interesting physical and chemical properties arise primarily from the electronic and/or magnetic interplay between the component metal ions. Among the extensive studies in the past two decades, those on lanthanide-containing clusters, lanthanide-exclusive or heterometallic with transition metal elements, are most notable. The research was driven by both the synthetic challenges for these generally elusive species and their intriguing magnetic properties, which are useful for the development of energy-efficient and environmentally friendly magnetic cooling technologies. Our efforts in this vein have been concentrated on developing rational synthetic methods for high-nuclearity lanthanide-containing clusters. By means of the now widely adopted approach of "ligand-controlled hydrolysis" of lanthanide ions, a great variety of cluster-type lanthanide hydroxide complexes had been prepared in the first half of this developing period (1999-2006). In this Account, our efforts since 2007 are summarized. These include (1) further development of synthetic strategies in order to expand the ligand scope and/or to increase the nuclearity (>25) of the cluster species and (2) magnetic studies pertinent to the pursuit of materials with a large magnetocaloric effect (MCE). Specifically, with the hope of expanding the family of ligands and producing clusters of previously unknown structures, we tested under hydrothermal or solvothermal conditions the use of readily available yet not commonly used ligands for controlling lanthanide hydrolysis; such ligands, carboxylates as mundane examples, tend to form insoluble complexes prior to any possible hydrolysis. We have also validated the use of preformed transition metal complexes as metalloligands for subsequent control of lanthanide hydrolysis toward heterometallic 3d-4f clusters. Furthermore, we demonstrated using ample examples that the presence of small anions as templates is essential to the assembly of high-nuclearity lanthanide-containing clusters and that maintaining a low concentration of the anion template(s) is a key to such success. It has been found that slow production/release of such anion templates by in situ ligand decomposition or absorption of atmospheric CO2 is effective in preventing precipitation of their lanthanide salts, allowing not only controllable lanthanide hydrolysis but also gradual and modular assembly of the giant cluster species. Magnetic studies targeting potential applications of such clusters as molecular magnetic coolers have also been conducted. The results are summarized in the second portion of this Account in an effort to establish a certain magneto-structure relationship. Of particular relevance is the possible correlation between MCE (evaluated using the isothermal magnetic entropy change, -ΔSM) and magnetic density, and the intracluster antiferromagnetic exchange coupling. We have also made some preliminary attempts at preparing processable and practically useful materials in the form of a monodisperse core-shell nanostructure. We succeeded in encapsulating a single nanosized heterometallic molecular cluster in a nanoshell of silica. It was found that such passivation not only helped stabilize the cluster but also reduced the magnetic interactions between individual clusters. These effects are reflected in the slightly enhanced value of -ΔSM for the core-shell composite over the parent unprotected cluster.

Rapid detection of drug resistance in mycobacterium tuberculosis using Tag Array chip

Objective To evaluate the sensitivity and specificity of Tag Array in detecting the clinical isolates of bone and joint tuberculosis. Methods Twenty-four strains of Mycobacterium tuberculosis having sequence differences in extracted plasmids of mutant strains. The plasmid was diluted into different concentrations, and then multiplex PCR amplification was performed to analyze the sensitivity and specificity of the chip system. A total of 187 clinical isolates were collected from patients with bone and joint tuberculosis. Among them, there were 50 strains of sensitive bacteria, and 137 strains of drug-resistant. Designed, optimized and prepared chip inspection system, sequencing and phenotypic drug susceptibility results were analyzed to evaluate the sensitivity and specificity of the gene chip. Results The mutants in accordance with sequencing result in the sets with 1×103copy/μl of template concentration and above; while 29 sets showed false negative result in the sets with 1×102 copy/μl. Among the 126 rifampicin phenotype drug resistance strains, 119 strains appeared mutation in rpoB gene. In the rifampicin phenotype sensitivity strains, 9 strains of rpoB gene showed mutation, the sensitivity was 94.40%, and specificity was 86.76%. Among the 118 isoniazid phenotype drug resistance strains, 109 strains appeared mutation in relevant drug resistance gene locus, the sensitivity was 92.37%, and specificity was 81.16%. Among the 44 ethambutol phenotype drug resistance strains, 27 strains appeared mutation in locus embB306, in the ethambutol phenotype sensitivity strains, the mutation in locus embB306 was detected in 6 strains, the sensitivity was 61.36%, and specificity was 95.80%. Among the 102 levofloxacin phenotype drug resistance strains, 81 strains appeared mutation in gyrA gene, 4 of the sensitivity strains were detected mutation in gyrA gene, the sensitivity was 79.41%, and specificity was 95.29%. Among the 112 streptomycin phenotype drug resistance strains, the chip detected 101 strains appeared mutation, the sensitivity of chip detection was 90.17%, and specificity was 84.00%. Since amikacin, capreomycin and kanamycin are in cross drug resistance, among the 67 phenotype drug resistance strains, 52 strains appeared mutation in relevant drug resistance gene locus, its sensitivity was 79.10% and specificity was 90.83%. Conclusion Tag Array chip can achieve rapid, accurate and high-throughput detection of tuberculosis resistance, which has important clinical significance and feasibility. Key words: Extensively drug-resistant tuberculosis; Genetic testing; Sensitivity and specificity

The structure heterogeneity of silica mesopores of Sba-15 in respect to the pluronic 123 template concentration

The analysis of structure heterogeneity factor of silica mesoporous SBA-15 has been conducted. The structure factor has been found to be different for low and high concentration of Pluronic-123 template. The structure heterogeneity of high concentration of Pluronic-123 has been found less than 1 while for low concentration, the structure heterogeneity was found to be larger than 1. This indicates the dissimilarity of the structure and can be used as a probe to detect the formation of large mesopores. It also was found that the system exhibits type IV and H1 adsorption type which indicates the capillary condensation and interconnected pores.

High Surface Area SOFC Electrode Materials Prepared at Traditional Sintering Temperatures

Solid oxide fuel cell (SOFC) electrode materials with surface areas up to 99 m2·g−1 were prepared at traditional sintering temperatures, 1050°C–1350°C, by sintering hybrid inorganic-organic materials in an inert atmosphere followed by calcination in air at 700°C. The electrode materials investigated were yttria-stabilized zirconia (YSZ), lanthanum strontium cobalt ferrite (LSCF), gadolinia doped ceria (GDC), and strontium titanate (STO). During sintering, an amorphous carbon template forms in situ and remains throughout the sintering process, aiding in the creation and preservation of mixed-metal-oxide nanomorphology. The carbon template is removed during subsequent calcination in air at 700°C, leaving behind a nanostructured ceramic. Phase stability, carbon template concentration, and specific surface area was determined for each mixed-metal-oxide. Final specific surface areas up to 83, 66, 95, and 99 m2·g−1 were achieved for YSZ, LSCF, GDC, and STO, respectively. The impact of high surface area YSZ on symmetrical YSZ-lanthanum strontium ferrite (LSF) cathode cell performance was evaluated in the temperature range of 550°C–800°C. Adding nanostructured YSZ decreased the electrochemical impedance by 45% at 550°C. The performance improvement lessened with increasing temperature, and at 800°C there was essentially no improvement. The findings reveal a promising approach to improving low temperature SOFC performance.

DNA Template Dilution Impacts Amplicon Sequencing-Based Estimates of Soil Fungal Diversity

Next generation sequencing of taxonomically relevant marker genes has enabled researchers to sample the richness, diversity, and composition of environmental microbiomes at previously unattainable depths. However, molecular methods may have unintended downstream consequences and the inadvertent undersampling of microbial communities may be a significant pitfall in microbiome profiling. One such procedure, dilution of the DNA template prior to polymerase chain reaction (PCR), may improve marker gene amplification by reducing chimeric read formation and decreasing PCR inhibitor concentrations. However, dilution unavoidably reduces target DNA template number per sample. We evaluated the effects of pre-PCR DNA template dilution on estimates of soil fungal microbiome diversity, composition, and species abundance distributions across a collection of 144 agricultural soil samples. Fungal DNA templates were serially diluted at 0-, 10-, 100-, and 1,000-fold and sequence data of diluted templates were compared with those of an identical set of undiluted templates. For three prairie soil samples, in addition to evaluating variation among replicates of individual samples, we serially diluted fungal DNA extracts from soil samples in triplicate and sequenced undiluted and diluted samples. DNA template dilution significantly reduced estimates of fungal richness and diversity, as compared with undiluted samples. Dilution of DNA template also resulted in reduced relative abundances of rare operational taxonomic units (OTUs) and increased relative abundances of common OTUs. Collectively, changes in OTU abundance distributions following dilution produced substantial shifts in overall fungal community composition. Our results highlight risks associated with sample dilution and point to the potential utility of quantifying pre-PCR template concentration in the estimation of microbiomes. We urge researchers to thoroughly document methods and to reconsider routine dilution of pre-PCR DNA templates particularly for low abundance microbiome samples. As efforts to profile environmental microbiomes using molecular sequencing approaches accelerate, developing an adequate understanding of potential methodological bottlenecks will increase our ability to accurately characterize and compare datasets.

Estimating and mitigating amplification bias in qualitative and quantitative arthropod metabarcoding

Amplicon based metabarcoding promises rapid and cost-efficient analyses of species composition. However, it is disputed whether abundance estimates can be derived from metabarcoding due to taxon specific PCR amplification biases. PCR-free approaches have been suggested to mitigate this problem, but come with considerable increases in workload and cost. Here, we analyze multilocus datasets of diverse arthropod communities, to evaluate whether amplification bias can be countered by (1) targeting loci with highly degenerate primers or conserved priming sites, (2) increasing PCR template concentration, (3) reducing PCR cycle number or (4) avoiding locus specific amplification by directly sequencing genomic DNA. Amplification bias is reduced considerably by degenerate primers or targeting amplicons with conserved priming sites. Surprisingly, a reduction of PCR cycles did not have a strong effect on amplification bias. The association of taxon abundance and read count was actually less predictable with fewer cycles. Even a complete exclusion of locus specific amplification did not exclude bias. Copy number variation of the target loci may be another explanation for read abundance differences between taxa, which would affect amplicon based and PCR free methods alike. As read abundance biases are taxon specific and predictable, the application of correction factors allows abundance estimates.

A rapid high-resolution melting method for differentiation of Leishmania species targeting lack gene

ObjectivesThe aim of this research is to verify that if lack gene can be used for differentiation of Leishmania under HRM assay. MethodsTwo specific primers were designed targeting polymorphic sites on the lack gene sequence. DNA from promastigotes of six species of Leishmania based on reference strains were tested following a HRM protocol. We also tested ten Chinese isolates in blind to validate our method. ResultsCombined with amplicon of the two primers, the six reference strains can be easily discriminated without the effect of initial concentration of DNA templates. Ten Chinese isolates detected by our HRM method resulted in full accord with the standard identification results in previous study. ConclusionHRM is a rapid and reproducible method to discriminate different Leishmania species and lack gene is a potential novel biological characteristic for easy differentiation of Leishmania isolates in China.

Molecular diagnosis Carpophilus dimidiatu (Coleoptera: Nitidulidae) based on species-specific PCR

Carpophilus dimidiatu (Fabricius), Carpophilus Pilosellus Motschulsky and Omosita colom (Linnaeus) are often found in food industry and stored products in China. C. dimidiatu is one common importance of nitidulid beetles pests. It is difficult to identify them only based on morphological characteristics. In this study, we describe species-specific PCR for the molecular identification of C. dimidiatu from C. Pilosellus and O. colon. Species-specific primer pairs were designed on the basis of variations in the mtDNA COI sequences. A 540bp-specific fragment of C. dimidiatu is generated by its species-specific prime pairs, whilst no such diagnostic bands were present in any of the two pest nitidulid beetles species tested. The results of the sensitivity test demonstrated that the detection limit of DNA template concentration was 0.5 ng/μL for C. dimidiatu. This study exhibited high specificity, reliability, rapid and low cost for C. dimidiatu diagnosis. Our data show that species-specific PCR is a powerful tool that can be used for the identification of nitidulid beetles pests.

Analysis of degraded casework DNA by redesigning a mini Y-STR multiplex

As partial genetic profile from degraded DNA samples of a case limiting the application of Y-STRs in forensic sciences, a new multiplex mini Y-STR typing was developed to obtain more useful information and complement current commercially available multiplex kits. The multiplex mini Y-STR amplification including three Y-STR loci (DYS385a/b, DYS389 II, and DYS392) was performed to generate amplicons smaller than those of PowerPlex® Y system over 140bp by redesigning primers. Allelic ladders for three mini Y-STR loci were created using a dilution of allelic ladders from the PowerPlex® Y system. A sensitivity test using serially diluted standard 9948 male DNA showed that three mini Y-STR loci were reliable at template concentrations as low as 0.25ng. By comparing of the multiplex mini Y-STR amplification and PowerPlex® Y system with referenced DNA samples, the results indicated stable concordance in genotyping. And then the successful analysis for the degraded DNA samples from the forensic case produced complete genetic profiles with these three loci. So the multiplex mini Y-STR typing combining the aforementioned commercially available kit increased chance of gaining of 16 Y-STR loci profiles within some core loci in Y Chromosome Haplotype Reference Database from forensic casework sample. It is an alternative strategy for recovering locus-specific information from degraded to redesign the suitable mini Y-STR complement besides new commercial Y-STR kits.

Evaluation of relative quantification of alternatively spliced transcripts using droplet digital PCR

IntroductionFor the relative quantification of isoform expression, RT-qPCR has been the gold standard for over a decade. More recently, digital PCR is becoming widely implemented, as it is promised to be more accurate, sensitive and less affected by inhibitors, without the need for standard curves. In this study we evaluated RT-qPCR versus RT-droplet digital PCR (ddPCR) for the relative quantification of isoforms in controls and carriers of the splice site mutation BRCA1 c.212+3A>G, associated with increased expression of several isoforms. Materials and methodsRNA was extracted from EBV cell lines of controls and heterozygous BRCA1 c.212+3A>G carriers. Transcript-specific plasmids were available to determine the efficiency, precision, reproducibility and accuracy of each method. ResultsBoth ddPCR and RT-qPCR were able to accurately quantify all targets and showed the same LOB, LOD and LOQ; also precision and reproducibility were similar. Both techniques have the same dynamic range and linearity at biologically relevant template concentrations. However, a significantly higher cost and workload was required for ddPCR experiments. ConclusionsOur study recognizes the potential and validity of digital PCR but shows the value of a highly optimized qPCR for the relative quantification of isoforms. Cost efficiency and simplicity turned out to be better for RT-qPCR.