Number Of Polymerase Chain Reaction Cycles Research Articles

First report of 'Candidatus Phytoplasma palmae' (16SrIV-A subgroup) associated with palm Lethal Yellowing disease on Cocos nucifera and Pritchardia sp. in Guadeloupe, French West Indies.

Lethal Yellowing (LY) disease causes major damage to palms in Central America and the Caribbean. It has been reported as far south as Antigua (Myrie et al., 2014). LY affects over forty palm species, seriously impacts the coconut industry and alters the landscapes on islands with a tourist-based economy. In March 2021, the presence of LY disease was regularly monitored in Guadeloupe. Two palm species (Cocos nucifera and Pritchardia sp.) died on a private property in Saint-Anne, Grande Terre. Yellowing of lower fronds and necrosis of inflorescences were reported on some neighboring palms. One symptomatic Cocos nucifera (GP21-007) and four symptomatic Pritchardia sp. (GP21-005, GP21-006, GP21-008 and GP21-009) were sampled by stem drilling. Samples from four asymptomatic coconut trees (GP21-001 to GP21-004) were collected in the locality of Deshaies. DNA was extracted from the nine sawdust samples following a cetyltrimethylammonium bromide (CTAB) modified protocol (Doyle and Doyle, 1990). A quantitative polymerase chain reaction (PCR), following the protocol described by Christensen et al. (2004), was performed on DNA to diagnose the presence of phytoplasmas. An exponential amplification was observed for all DNA extracts from symptomatic palm samples (threshold number of PCR cycles (Ct) ranged from 18.50 to 23.58). DNA from asymptomatic samples yielded negative results (undetermined Ct). To identify the phytoplasma associated with LY, DNA samples were subjected to PCR, based on the 16SrRNA gene, plus internal transcribed spacers (ITS) using P1-1T (Pilet et al., 2021)/P7 (Schneider et al., 1995) primers, and secA gene using the primer pair secAFor1/secARev1 (Hodgetts et al. 2008). Amplicons of 1.8 kb covering the 16S ribosomal operon and 830 bp for the secA gene were produced using DNA from symptomatic trees. All amplicons were double strand sequenced (Genewiz, UK). The corresponding sequences were deposited in GenBank and subjected to BLASTn on NCBI. Sequences of the ribosomal operon gene (accession no. ON521114 to ON521118) were identical for the five positive samples. Sequencing revealed two distinct ribosomal operons with heterozygous peaks on the DNA chromatogram. The first aMino ambiguity (M = Adenine or Cytosine) was observed in the 16Sr RNA gene. The second was observed in the first intergenic transcript spacer. The 16S rDNA sequence (M = Cytosine) presented 100% identity with accession no. HQ613874 and 99.93% with accession no. U18747, the reference sequence for 'Candidatus Phytoplasma palmae'. The virtual RFLP pattern derived from the 16S rDNA F2nR2 fragment and identified using iPhyclassifier (Zhao et al. 2009) was identical to the reference pattern for the 16SrIV-A subgroup. A unique sequence was obtained for the partial secA gene (OP136139 to OP136143), sharing 100% identity with EU267187 for the palm LY phytoplasma preprotein translocase subunit (secA) gene. This is the first report of 'Ca. Phytoplasma palmae' (subgroup 16SrIV-A) associated with palm LY disease on Cocos nucifera and Pritchardia sp. in Guadeloupe. Measures to eradicate LY were implemented as soon as its presence was confirmed in Guadeloupe. LY phytoplasmas continue to spread in the Caribbean and are approaching South America, where the known vector, Haplaxius crudus, has already been reported (Silva et al., 2019). This poses a major threat to the coconut economy and the diversity of palm trees.

A novel ADA-coated UCNPs@NB sensing platform combined with nucleic acid amplification for rapid detection of Escherichia coli

In this study, we reported a novel sensing platform based on fluorescence quenching composed of alendronic acid (ADA) coated upconversion nanoparticles (UCNPs) and Nile Blue (NB) combined with polymerase chain reaction (PCR) for rapid, sensitive, and specific detection of Escherichia coli (E. coli). As a fluorescence acceptor, NB has a broad absorption band and can quench upconversion fluorescence intensity at 544 nm and 658 nm based on IFE. PCR is a double-stranded DNA (dsDNA) amplification technique with high specificity. The NB-dsDNA complex can be formed by intercalation of NB between base pairs and groove of dsDNA, leading to upconversion fluorescence recovery. The ADA-coated UCNPs@NB sensing platform achieved to detect E. coli in 1.5 h, with a lower limit of detection (33 CFU mL−1). In addition, the sensitivity of the ADA@UCNPs-NB fluorescence sensor under different PCR cycle numbers was discussed. The results showed that the proposed sensor could effectively shorten the assay time (1.0 h) while maintaining excellent sensitivity. This study demonstrated a rapid and sensitive analytical method for detecting E. coli in chicken, providing a reference for constructing PCR fluorescence sensors.

Real-time PCR assays to detect 10 Shiga toxin subtype (Stx1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g) genes

To develop subtyping methods for Shiga toxin (Stx)1a, Stx1c, Stx1d, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, and Stx2g genes for epidemiological analyses of Shiga toxin-producing Escherichia coli (STEC), we developed 10 simplex real-time polymerase chain reaction (PCR) assays with reference to 284 valid stx sequences and evaluated their specificity and quantitative accuracy using STEC and non-STEC isolates and recombinant plasmids, respectively. Three stx1 and 5 stx2 subtype genes, except for stx2c and stx2d, were detected with high specificity using STEC isolates. However, some stx2a sequences potentially being close to both Stx2a and Stx2d cluster in neighbor-joining cluster analysis were positive for stx2a and stx2d by real-time PCR. For the stx2c assay, the number of real-time PCR cycles was reduced to avoid unnecessary false-positive results. Based on these considerations, the real-time PCR assays developed here might aid epidemiological investigations of infections or outbreaks caused by STEC harboring any of the stx subtype genes.

Clinically relevant variation in FLT3-ITD quantitation as a result of PCR cycle number and ITD insertion size

FLT3 internal tandem duplication (ITD) quantitation is key to prognostication in acute myeloid leukaemia (AML). One potential source of variability in the allelic ratio (AR) is the number of polymerase chain reaction (PCR) cycles used. Using 30 archived samples of varying ITD lengths and AR, we compared two FLT3-ITD assays (Huang and RATIFY), evaluated the effect of PCR cycle number on each assay, and examined the potential clinical consequences. Huang and RATIFY assays at 35 and 27 PCR cycles, respectively, were highly concordant. A progressive decrease in AR (median 47%) was observed with the RATIFY assay when the PCR cycles were increased from 27 to 35 cycles, potentially impacting risk categorisation in 29% of patients. In contrast, minimal change in AR was observed with the Huang assay. Hence, both FLT3-ITD assays were almost identical using respective standard conditions, but the effect of PCR cycle number is assay-dependent, which may impact risk stratification in AML.

Cerebrospinal Fluid Bacillary Load by Xpert MTB/RIF Ultra Polymerase Chain Reaction Cycle Threshold Value Predicts 2-Week Mortality in Human Immunodeficiency Virus-Associated Tuberculous Meningitis.

BackgroundThe World Health Organization recommends GeneXpert MTB/RIF Ultra (Xpert Ultra), a fully automated polymerase chain reaction (PCR) assay, as the initial tuberculous meningitis (TBM) diagnostic test. The assay’s PCR cycle threshold (Ct) values represent the number of PCR cycles required for probe signal to be detected (low Ct value = high bacillary load) and may approximate tuberculosis (TB) bacillary load. We measured the relationship between cerebrospinal fluid (CSF) TB bacillary load with mortality. MethodsWe prospectively enrolled 102 human immunodeficiency virus (HIV)–positive Ugandans with probable or definite TBM from April 2015 to August 2019. Xpert Ultra Ct tertiles and semi-quantitative categories were separately analyzed as predictors of 2-week mortality. We investigated associations between Ct and baseline clinical and CSF parameters. ResultsSubjects with Ct values in the low tertile (ie, high bacillary load) had 57% 2-week mortality—worse than the intermediate (17%) and high (25%) Ct tertiles and Xpert Ultra–negative (30%) probable TBM cases (P = .01). In contrast, the reported semi-quantitative Xpert Ultra categorization was less precise; with the medium to low category trending toward worse 2-week survival (42%) compared with very low (28%), trace (26%), and negative (30%) categories (P = .48). Ct tertile was significantly associated with baseline CSF lactate (P = .03). ConclusionsHigh CSF TB bacillary load, as measured by Xpert Ultra Ct tertile, is associated with an almost 2-fold higher 2-week mortality in HIV-associated TBM and is a better predictor than the reported Xpert Ultra semi-quantitative category. Xpert Ultra Ct values could identify TBM patients at increased risk of death who may benefit from enhanced supportive care.

Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers

BackgroundRNA-seq and small RNA-seq are powerful, quantitative tools to study gene regulation and function. Common high-throughput sequencing methods rely on polymerase chain reaction (PCR) to expand the starting material, but not every molecule amplifies equally, causing some to be overrepresented. Unique molecular identifiers (UMIs) can be used to distinguish undesirable PCR duplicates derived from a single molecule and identical but biologically meaningful reads from different molecules.ResultsWe have incorporated UMIs into RNA-seq and small RNA-seq protocols and developed tools to analyze the resulting data. Our UMIs contain stretches of random nucleotides whose lengths sufficiently capture diverse molecule species in both RNA-seq and small RNA-seq libraries generated from mouse testis. Our approach yields high-quality data while allowing unique tagging of all molecules in high-depth libraries.ConclusionsUsing simulated and real datasets, we demonstrate that our methods increase the reproducibility of RNA-seq and small RNA-seq data. Notably, we find that the amount of starting material and sequencing depth, but not the number of PCR cycles, determine PCR duplicate frequency. Finally, we show that computational removal of PCR duplicates based only on their mapping coordinates introduces substantial bias into data analysis.

Evaluating Established Methods for Rumen 16S rRNA Amplicon Sequencing With Mock Microbial Populations.

The rumen microbiome scientific community has utilized amplicon sequencing as an aid in identifying potential community compositional trends that could be used as an estimation of various production and performance traits including methane emission, animal protein production efficiency, and ruminant health status. In order to translate rumen microbiome studies into executable application, there is a need for experimental and analytical concordance within the community. The objective of this study was to assess these factors in relation to selected currently established methods for 16S phylogenetic community analysis on a microbial community standard (MC) and a DNA standard (DS; ZymoBIOMICSTM). DNA was extracted from MC using the RBBC method commonly used for microbial DNA extraction from rumen digesta samples. 16S rRNA amplicon libraries were generated for the MC and DS using primers routinely used for rumen bacterial and archaeal community analysis. The primers targeted the V4 and V3–V4 region of the 16S rRNA gene and samples were subjected to both 20 and 28 polymerase chain reaction (PCR) cycles under identical cycle conditions. Sequencing was conducted using the Illumina MiSeq platform. As the bacteria contained in the microbial mock community were well-classified species, and for ease of explanation, we used the results of the Basic Local Alignment Search Tool classification to assess the DNA, PCR cycle number, and primer type. Sequence classification methodology was assessed independently. Spearman’s correlation analysis indicated that utilizing the repeated bead beating and column method for DNA extraction in combination with primers targeting the 16S rRNA gene using 20 first-round PCR cycles was sufficient for amplicon sequencing to generate a relatively accurate depiction of the bacterial communities present in rumen samples. These results also emphasize the requirement to develop and utilize positive mock community controls for all rumen microbiomic studies in order to discern errors which may arise at any step during a next-generation sequencing protocol.

Assessment of the most reliable sites in mandibular bone for the best deoxyribonucleic acid yield for expeditive human identification in forensics.

Background:In recent years, the techniques used to identify human remains post accidents, trauma or in case of criminal investigation have been expanded, improved and rendered more complex by the emergence of technologies based on deoxyribonucleic acid (DNA) analysis. In the head and neck area, tooth has been proven to be the best quantitative source for DNA but in certain cases where the mandible specimen is edentulous or the tooth is extensively destroyed with caries, large dental restorations, mobile, or if they show any perimortem or postmortem fractures, sampling of such tooth specimen is usually avoided. In such situations, bone is considered the next best site for DNA analysis. Mandible being the largest, strongest and dense cortical bone is the most prominent facial bone that can be easily disarticulated. It can be analyzed for the best short tandem repeat (STR) segment qualitative amplification using polymerase chain reaction (PCR) technique for forensic analysis which can be used for gender and age determination.Aim:The aim of this study is to determine the best site for optimum quantitative and qualitative yield of DNA for amplification using specific and standard STR segment by conventional PCR technique.Methodology:Fifteen mandibular samples exposed to different environmental conditions were collected. Bone pieces of 1 cm × 1 cm were cut from each mandible from three sites, i.e., the ramus, angle and body, wherein the genomic DNA was isolated and was subjected to PCR using restricted number of 25 cycles.Results:The STR segment D3S1358 from clone RP11-438F9 used for the study showed very good amplification in restricted number of PCR cycles in the ramus region with number of repeats in every 15th genomic region.Conclusion:This study highlights the use of mandibular bone for the expeditive human identification. As per the study, the ramus of the mandible gave high quantitative and qualitative yield of DNA with thick amplification band of the STR segment as compared to the body and angle of the mandible. Thus ramus of the mandible can be preferred over other sites for molecular forensic investigations.

Diversity analysis of sulfite- and sulfate-reducing microorganisms by multiplex dsrA and dsrB amplicon sequencing using new primers and mock community-optimized bioinformatics.

Genes encoding dissimilatory sulfite reductase (DsrAB) are commonly used as diagnostic markers in ecological studies of sulfite- and sulfate-reducing microorganisms. Here, we developed new high-coverage primer sets for generation of reductive bacterial-type dsrA and dsrB polymerase chain reaction (PCR) products for highly parallel amplicon sequencing and a bioinformatics workflow for processing and taxonomic classification of short dsrA and dsrB reads. We employed two diverse mock communities that consisted of 45 or 90 known dsrAB sequences derived from environmental clones to precisely evaluate the performance of individual steps of our amplicon sequencing approach on the Illumina MiSeq platform. Although PCR cycle number, gene-specific primer mismatches and stringent filtering for high-quality sequences had notable effects on the observed dsrA and dsrB community structures, recovery of most mock community sequences was generally proportional to their relative input abundances. Successful dsrA and dsrB diversity analysis in selected environmental samples further proved that the multiplex amplicon sequencing approach is adequate for monitoring spatial distribution and temporal abundance dynamics of dsrAB-containing microorganisms. Although tested for reductive bacterial-type dsrAB, this method is readily applicable for oxidative-type dsrAB of sulfur-oxidizing bacteria and also provides guidance for processing short amplicon reads of other functional genes.

Factors affecting peak height variability for short tandem repeat data

In forensic DNA analysis a DNA extract is amplified using polymerase chain reaction (PCR), separated using capillary electrophoresis and the resulting DNA products are detected using fluorescence. Sampling variation occurs when the DNA molecules are aliquotted during the PCR setup stage and this translates to variability in peak heights in the resultant electropherogram or between electropherograms generated from a DNA extract. Beyond the variability caused by sampling variation it has been observed that there are factors in generating the DNA profile that can contribute to the magnitude of variability observed, most notably the number of PCR cycles. In this study we investigate a number of factors in the generation of a DNA profile to determine which contribute to levels of peak height variability.

Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations

The TaqMan probes that have been long and effectively used in real-time polymerase chain reaction (PCR) may also be used in DNA melting analysis. We studied some factors affecting efficiency of the approach such as (i) number of asymmetric PCR cycles preceding DNA melting analysis, (ii) choice of fluorophores for the multiplex DNA melting analysis, and (iii) choice of sense or antisense TaqMan probes for optimal resolution of wild-type and mutant alleles. We also determined ΔTm (i.e., the temperature shift of a heteroduplex relative to the corresponding homoduplex) as a means of preliminary identification of mutation type. In experiments with serial dilution of mutant KRAS DNA with wild-type DNA, the limit of detection of mutant alleles was 1.5–3.0%. Using DNA from both tumor and formalin-fixed paraffin-embedded tissues, we demonstrated a high efficiency of TaqMan probes in mono- and multiplex mutation scanning of KRAS, NRAS (codons 12, 13, and 61), and BRAF (codon 600) genes. This cost-effective method, which can be applied to practically any mutation hot spot in the human genome, combines simplicity, ease of execution, and high sensitivity—all of the qualities required for clinical genotyping.

Electrochemical magnetoassay coupled to PCR as a quantitative approach to detect the soybean transgenic event GTS40-3-2 in foods

Simple, cost-effective and reliable tools for the quantification of genetically modified organisms (GMO) in food and feed are highly demanded to enforce labelling legislation in the EU. Herein, we report a novel method for quantitative analysis of genetically modified soybean with the event GTS-40-3-2, also known as Roundup Ready (RR) soybean, using magnetoassays with electrochemical detection, coupled to DNA amplification by end-point polymerase chain reaction (PCR). For the proposed work, two DNA sequences were targeted via hybridisation onto magnetic beads, one specific for the transgenic event and the other for the taxon or species-specific lectin gene. Enzymatic labelling was performed to obtain an electrochemically active product measured by chronoamperometry. By optimising the number of PCR cycles, among other parameters, two magnetoassays coupled to PCR were successfully accomplished and linearity was obtained in the ranges of 53–4425 and 1093–88,496 DNA copies for the event-specific and lectin sequences, respectively. The proposed method provides accurate and precise RR soybean quantitative results, being effectively compared to those obtained by real-time PCR, as the reference method. These findings confirm the suitability of the method as an alternative tool for GMO quantification.

Characterization of new EST-linked microsatellites in the rough periwinkle (Littorina saxatilis) and application for parentage analysis

The rough periwinkle, Littorina saxatilis (Olivi, 1792), has become a model of choice for studying processes of local adaptation and ecological speciation (e.g. Johannesson et al., 2010). Interestingly, this ovoviviparous intertidal snail displays extreme female promiscuity, since a female can carry offspring sired by at least 23 males (Panova et al., 2010). Microsatellite-based parentage analysis has proved valuable for the study of multiple paternity in L. saxatilis (Makinen, Panova & Andre, 2007; Panova et al., 2010). However, three of the five markers used in these two previous studies have shown problems such as heterozygote deficiencies, null alleles and variation in allele size not equal to repeat motif (Panova et al., 2008). In many invertebrates, including molluscs in general and L. saxatilis in particular, microsatellite flanking regions contain indels and mutations causing amplification failures (null alleles), as well as cryptic repetitive DNA leading to multiple bands in polymerase chain reaction (PCR) products (McInerney et al., 2011). Such issues may significantly affect the accuracy of parentage assignment by increasing the rate of genotyping errors. To avoid these problems, the present study aimed at developing expressed sequence tag (EST)-linked microsatellites, which are likely to have more conserved flanking regions (Bouck & Vision, 2007). Six new EST-linked microsatellites were developed and combined with two recently-published loci (McInerney et al., 2009), and the performance of this new set of markers was tested for parentage analysis. EST sequences were downloaded from the Littorina Sequence Database (Canback et al., 2012), and microsatellites were identified with Tandem Repeats Finder (Benson, 1999). The 57 best di-, trior tetranucleotide microsatellites, displaying a minimum of eight repeats, a minimum of 75% matches and sufficiently long flanking regions (.40 bp), were loaded in Geneious Pro v. 5.1.7 (Drummond et al., 2010; Biomatters Ltd), and the Primer3 plugin (Rozen & Skaletsky, 2000) was used to design primer pairs with the following parameters: optimal primer length 1⁄4 20 bp (range 1⁄4 18–25 bp), optimal GC content 1⁄4 50% (range 1⁄4 30–80%), optimal Tm 1⁄4 608C (range 1⁄4 55– 618C) with a Tm difference not larger than 28C between the forward and reverse primers from the same pair and product length 1⁄4 100–400 bp. Finally, 36 primer pairs were selected for further PCR amplification tests. A ‘universal tail’ was added to the 50-end of each forward primer to reduce the genotyping cost (Schuelke, 2000). Three distinct ‘universal tails’ were used (tail1, tail2 or tail4), as described by Real, Schmidt & Hughes (2009). A PIG-tail (50-GTTTCTT) was also added to the 50-end of each reverse primer to avoid ‘plus-A’ PCR artefacts (Brownstein et al., 1996). In addition, three microsatellites developed by McInerney et al. (2009), with perfect trinucleotide (Lsax18 and Lsax20) or tetranucleotide repeats (Lsax13), were included in the study. All 39 microsatellites were tested with a population sample of L. saxatilis collected on the western Swedish coast, on the island of Salto (588530N, 118100E). Individuals were stored in ethanol at 2208C. DNA was extracted using the EZNA Mollusc DNA Extraction kit following the manufacturer’s instructions. Amplification tests were performed on eight individuals for all 39 loci. Each EST-linked microsatellite was amplified by PCR in a 10-ml volume, containing 1 RBC reaction buffer (RBC Bioscience), 1.0–2.0 mM MgCl2, 0.1 mM each dNTP, 0.125 mM ‘universal’ primer end-labelled with a WellRED dye (D2, D3 or D4; Sigma-Aldrich), 0.0125 mM tailed forward primer, 0.125 mM reverse primer, 0.025 U ml RBC Taq DNA polymerase (RBC Bioscience) and 1 ml DNA template. The three microsatellites published by McInerney et al. (2009) were amplified following the same protocol as above but with 0.15 mM forward primer end-labelled with a WellRED dye (Sigma-Aldrich) and 0.15 mM reverse primer. PCR amplifications were performed in a Veriti Thermal Cycler (Life Technologies). A touchdown procedure was incorporated in the thermal cycling regime to increase the stringency of the PCRs: 948C for 3 min, Ta1 for 2 min, 728C for 1 min, (948C for 30 s, Ta1 218C for 30 s [218C per cycle until Ta2], 728C for 1 min) 9–12 cycles, (948C for 30 s, Ta2 for 30 s, 728C for 1 min) 22–30 cycles, 728C for 5 min. A total of 35 PCR cycles were run by default, but this was increased to 40 cycles when the peak intensity was too low. Details of the PCR protocol, i.e. MgCl2 concentration, number of PCR cycles and type of ‘universal’ tail, are provided in Table 1 for the successfully amplified loci. Amplification success was checked with electrophoresis on 1% agarose gel in 0.5 tris-borate-EDTA. Finally, PCR products were run on a Beckman Coulter CEQ 8000 capillary sequencer

Biases for detecting arbuscular mycorrhizal fungal mixture by terminal restriction fragment length polymorphism (T-RFLP)

Terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified ribosomal RNA genes is used for profiling microbial communities and sometimes for species richness and relative abundance estimation in environmental samples. However, the T-RFLP fingerprint may be subject to biases during the procedure, influencing the detection of real community structures in the environment. To investigate possible sources of T-RFLP bias, 18S rRNA gene clones derived from two arbuscular mycorrhizal fungal sequences were combined in simple pairwise mixes to assess the effects of polymerase chain reaction cycle number, plant genomic DNA purification method and varying template ratio on the template-to-product ratio as measured by relative T-RF peak area. Varying cycle numbers indicated that amplification was still in the exponential phase at the cycle numbers lower than 18, so these small cycle numbers were used for the comparison of template-to-product quantities. Relative abundance estimated from T-RF peak ratios varied with different purification procedures, but the best results, closest to input ratios, were obtained by using phenol-chloroform purification. The presence of an excess of unpurified non-target plant genomic DNA generated a bias towards lower or overestimation of relative abundance. We conclude that a low number of amplification cycles and stringent DNA purification are necessary for accurate mixed sample analysis by T-RFLP.

Nanoscale Superstructures Assembled by Polymerase Chain Reaction (PCR): Programmable Construction, Structural Diversity, and Emerging Applications

Polymerase chain reaction (PCR) is an essential tool in biotechnology laboratories and is becoming increasingly important in other areas of research. Extensive data obtained over the last 12 years has shown that the combination of PCR with nanoscale dispersions can resolve issues in the preparation DNA-based materials that include both inorganic and organic nanoscale components. Unlike conventional DNA hybridization and antibody-antigen complexes, PCR provides a new, effective assembly platform that both increases the yield of DNA-based nanomaterials and allows researchers to program and control assembly with predesigned parameters including those assisted and automated by computers. As a result, this method allows researchers to optimize to the combinatorial selection of the DNA strands for their nanoparticle conjugates. We have developed a PCR approach for producing various nanoscale assemblies including organic motifs such as small molecules, macromolecules, and inorganic building blocks, such as nanorods (NRs), metal, semiconductor, and magnetic nanoparticles (NPs). We start with a nanoscale primer and then modify that building block using the automated steps of PCR-based assembly including initialization, denaturation, annealing, extension, final elongation, and final hold. The intermediate steps of denaturation, annealing, and extension are cyclic, and we use computer control so that the assembled superstructures reach their predetermined complexity. The structures assembled using a small number of PCR cycles show a lower polydispersity than similar discrete structures obtained by direct hybridization between the nanoscale building blocks. Using different building blocks, we assembled the following structural motifs by PCR: (1) discrete nanostructures (NP dimers, NP multimers including trimers, pyramids, tetramers or hexamers, etc.), (2) branched NP superstructures and heterochains, (3) NP satellite-like superstructures, (4) Y-shaped nanostructures and DNA networks, (5) protein-DNA co-assembly structures, and (6) DNA block copolymers including trimers and pentamers. These results affirm that this method can produce a variety of chemical structures and in yields that are tunable. Using PCR-based preparation of DNA-bridged nanostructures, we can program the assembly of the nanoscale blocks through the adjustment of the primer intensity on the assembled units, the number of PCR cycles, or both. The resulting structures are highly complex and diverse and have interesting dynamics and collective properties. Potential applications of these materials include chirooptical materials, probe fabrication, and environmental and biomedical sensors.

Alternating Plasmonic Nanoparticle Heterochains Made by Polymerase Chain Reaction and Their Optical Properties.

Organization of nanoparticles (NPs) of different materials into superstructures of higher complexity represents a key challenge in nanotechnology. Polymerase chain reaction (PCR) was used in this study to fabricate chains consisting of plasmonic NPs of different sizes, thus denoted heterochains. The NPs in such chains are connected by DNA oligomers, alternating in a sequence big-small-big-small-... and spanning lengths in the range of 40-300 nm by varying the number of PCR cycles. They display strong plasmonic chirality at 500-600 nm, the chiral activity revealing nonmonotonous dependence on the length of heterochains. We find the strength of surface-enhanced Raman scattering (SERS) to increase with chain length, while the chiral response initially increased and then decreased with the number of PCR cycles. The relationship between the optical properties of the heterochains and their structure/length is discussed. The length-dependent intense optical response of the plasmonic NP heterochains holds great potential for biosensing applications.

Effects of OTU Clustering and PCR Artifacts on Microbial Diversity Estimates

Next-generation sequencing has increased the coverage of microbial diversity surveys by orders of magnitude, but differentiating artifacts from rare environmental sequences remains a challenge. Clustering 16S rRNA sequences into operational taxonomic units (OTUs) organizes sequence data into groups of 97 % identity, helping to reduce data volumes and avoid analyzing sequencing artifacts by grouping them with real sequences. Here, we analyze sequence abundance distributions across environmental samples and show that 16S rRNA sequences of >99 % identity can represent functionally distinct microorganisms, rendering OTU clustering problematic when the goal is an accurate analysis of organism distribution. Strict postsequencing quality control (QC) filters eliminated the most prevalent artifacts without clustering. Further experiments proved that DNA polymerase errors in polymerase chain reaction (PCR) generate a significant number of substitution errors, most of which pass QC filters. Based on our findings, we recommend minimizing the number of PCR cycles in DNA library preparation and applying strict postsequencing QC filters to reduce the most prevalent artifacts while maintaining a high level of accuracy in diversity estimates. We further recommend correlating rare and abundant sequences across environmental samples, rather than clustering into OTUs, to identify remaining sequence artifacts without losing the resolution afforded by high-throughput sequencing.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

Asymmetric and symmetric PCR of gold nanoparticles: A pathway to scaled-up self-assembly with tunable chirality

Scalable preparation of nanoparticle assemblies and superstructures with control over the size, composition, and geometry represents a challenge for many practical applications of nanomaterials. DNA nanotechnology offers both versatility of particle arrangements and multiple gateways to interesting optical, electronic, biological, and catalytic properties. In this work we used a polymerase chain reaction (PCR) to produce complex superstructures from heterogeneously-sized gold nanoparticles. By controlling the number of PCR cycles and primer density, two distinct superstructures were assembled at asymmetric and symmetric PCR. With the increasing of PCR number, the size of structures was accordingly increased in the range 35–600nm. More importantly, the scaled-up GNP superstructures exhibited tunable chirality, which showed distinct differences between asymmetric and symmetric PCR. The origin of the chirality was analyzed from both an experimental and theoretical point of view. The heterogeneity of GNPs and conformational transition of self-assembly contributed to the occurrence of chirality in the GNP superstructures. PCR could be a very important and potentially useful tool for the controllable preparation of scaled-up superstructures with tunable chirality, and this experimental and mechanical research could significantly promote the technical development of chiral material preparation.

Claudin-11 expression increased in spermatogenic defect in human testes

Expression of claudin-11, which builds up blood-testis barrier (BTB), was increased in decreased spermatogenesis, including in hypospermatogenesis, spermatocytic and maturation arrest, and Sertoli cell only (SCO) testes. Increased claudin-11 immunoreactivity was observed at the inter-Sertoli tight junctions in maturation arrest and in the cytoplasm of Sertoli cells in SCO. The late spermatogenic wave may negatively regulate claudin-11 gene activation and the subcellular localization of claudin-11 in Sertoli cells, thus altering the BTB in the human testis.