Naked-eye detection of specific DNA sequences amplified by the polymerase chain reaction with nanocomposite beads.

We developed a novel nanocomposite bead system for detection by the naked eye of specific DNA sequences amplified by the polymerase chain reaction (PCR). The DNA probes, which were complementary to the target DNA, are conjugated with the nanocomposite beads. If the amplified products contained sequences complementary to the probes, the beads aggregated through sandwich hybridization. The aggregation was detectable as precipitation of the nanocomposite beads. The results were determined visually and did not require instrumental detection. The assay was sensitive enough to detect PCR products with a detection limit of 10 copies/tube for DNA templates. This technique is that all needed components are included within the initial cap, so that the risk of carryover contamination is very low. The nanocomposite bead system has broad application prospects for the detection of specific DNA sequences in biological and biomedical research.

Possible steps of complete disassembly of post-termination complex by yeast eEF3 deduced from inhibition by translocation inhibitors.

Ribosomes, after one round of translation, must be recycled so that the next round of translation can occur. Complete disassembly of post-termination ribosomal complex (PoTC) in yeast for the recycling consists of three reactions: release of tRNA, release of mRNA and splitting of ribosomes, catalyzed by eukaryotic elongation factor 3 (eEF3) and ATP. Here, we show that translocation inhibitors cycloheximide and lactimidomycin inhibited all three reactions. Cycloheximide is a non-competitive inhibitor of both eEF3 and ATP. The inhibition was observed regardless of the way PoTC was prepared with either release factors or puromycin. Paromomycin not only inhibited all three reactions but also re-associated yeast ribosomal subunits. On the other hand, sordarin or fusidic acid, when applied together with eEF2/GTP, specifically inhibited ribosome splitting without blocking of tRNA/mRNA release. From these inhibitor studies, we propose that, in accordance with eEF3's known function in elongation, the release of tRNA via exit site occurs first, then mRNA is released, followed by the splitting of ribosomes during the disassembly of post-termination complexes catalyzed by eEF3 and ATP.

Ribosome recycling step in yeast cytoplasmic protein synthesis is catalyzed by eEF3 and ATP.

After each round of protein biosynthesis, the posttermination complex (PoTC) consisting of a ribosome, mRNA, and tRNA must be disassembled into its components for a new round of translation. Here, we show that a Saccharomyces cerevisiae model PoTC was disassembled by ATP and eukaryotic elongation factor 3 (eEF3). GTP or ITP functioned with less efficiency and adenosine 5gamma'-(beta,gamma-imido)triphosphate did not function at all. The k(cat) of eEF3 was 1.12 min(-1), which is comparable to that of the in vitro initiation step. The disassembly reaction was inhibited by aminoglycosides and cycloheximide. The subunits formed from the yeast model PoTC remained separated under ionic conditions close to those existing in vivo, suggesting that they are ready to enter the initiation process. Based on our experimental techniques used in this paper, the release of mRNA and tRNA and ribosome dissociation took place simultaneously. No 40S*mRNA complex was observed, indicating that eEF3 action promotes ribosome recycling, not reinitiation.

Quantitative detection of chloroethene-reductive bacteria Dehalococcoides spp. using alternately binding probe competitive Polymerase Chain Reaction.

Dehalococcoides spp. are responsible for the reductive dehalogenation of environmental contaminants and are candidates for engineered bioremediation. The development of a sensitive, reliable, and rapid method for the quantification of Dehalococcoides spp. is required for the effective use of the organisms in bioremediation sites. Here, we describe the quantification of the 16S rRNA gene of Dehalococcoides spp. using a recently developed quantification method named alternately binding probe competitive PCR (ABC-PCR). The primers and probe sets that were newly designed for ABC-PCR were found to have a high specificity for Dehalococcoides spp. The standard curve of ABC-PCR had a good fitting (R = 0.999), and the lower detection limit was 10 copies/microl of template DNA. We also investigated the effects of inherent PCR-inhibiting compounds in an environmental sample on the quantification using ABC-PCR or real-time PCR by adding the soil extraction solution to PCR mixtures. ABC-PCR was more robust against the PCR amplification inhibitors than real-time PCR. The copy number of the 16S rRNA gene of Dehalococcoides spp. in soil and groundwater samples was successfully quantified using ABC-PCR. In conclusion, ABC-PCR is useful for the quantification of Dehalococcoides spp. populations and dynamics at bioremediation sites.

Universal quenching probe system: flexible, specific, and cost-effective real-time polymerase chain reaction method.

We have developed a flexible, specific, and cost-effective real-time polymerase chain reaction (PCR) method. In this technique, a quenching probe (QProbe) and a nonfluorescent 3'-tailed probe are used. The QProbe is a singly labeled oligonucleotide bearing a fluorescent dye that is quenched via electron transfer between the dye and a guanine base at a particular position. The nonfluorescent 3'-tailed probe consists of two parts: one is the target-specific sequence on the 5' side, and the other is complementary to the QProbe on the 3' side. When the QProbe/nonfluorescent 3'-tailed probe complex hybridizes with the target in PCR, the fluorescence of the dye is quenched. Fluorescence quenching efficiency is proportional to the amount of the target. We called this method the universal QProbe system. This method substantially reduces the cost of real-time PCR setup because the same QProbe can be used for different target sequences. Moreover, this method allows accurate quantification even in the presence of nonspecific PCR products because the use of nonfluorescent 3'-tailed probe significantly increases specificity. Our results demonstrate that this method can accurately and reproducibly quantify specific nucleic acid sequences in crude samples, comparable with conventional TaqMan chemistry. Furthermore, this method is also applicable to single-nucleotide polymorphism (SNP) genotyping.

Real-time reverse transcription loop-mediated isothermal amplification for rapid and simple quantification of WT1 mRNA.

OBJECTIVES: This study developed a novel MRD monitoring method targeting Wilms' tumor gene (WT1) mRNA using reverse transcription loop-mediated isothermal amplification (RT-LAMP). DESIGN AND METHODS: A primer set for the assay was designed on the basis of the sequences between the 17AA and KTS regions of WT1mRNA. WT1 mRNA was quantified by real-time RT-LAMP and the accuracy of RT-LAMP was compared with that of real-time RT-PCR. RESULTS: The standard curve was expressed as a linear relationship between the log copy numbers of WT1 mRNA ranging from 6.8 x 10 to 6.8 x 10(9) copies and the threshold time with a correlation coefficient of R(2) > 0.994. The measured values obtained by RT-LAMP strongly correlated with those obtained by real-time RT-PCR. CONCLUSION: RT-LAMP can be used to determine WT1 mRNA expression levels. This assay will contribute to a more specific, simple, and rapid MRD monitoring than conventional assays.

Chemical synthesis and properties of 5-taurinomethyluridine and 5-taurinomethyl-2-thiouridine.

Unique taurine-containing uridine derivatives, 5-taurinomethyluridine (tau m5U) and 5-taurinomethyl-2-thiouridine (tau m5s2U), which were discovered in mammalian mitochondrial tRNAs, exist at the first position of the anticodon. In this paper, we report the first efficient synthesis of tau m5U and tau m5s2U and describe their physicochemical properties. These modified ribonucleosides were synthesized by the reaction of 5-substituted uridine derivatives with a tetrabutylammonium salt of taurine that is highly reactive and well-soluble in common organic solvents. UV and 1H NMR spectrometric studies revealed the structural properties of the taurine-containing base moieties and the sugar conformations of these modified ribonucleosides.

Modified uridines with C5-methylene substituents at the first position of the tRNA anticodon stabilize U.G wobble pairing during decoding.

Post-transcriptional modifications at the first (wobble) position of the tRNA anticodon participate in precise decoding of the genetic code. To decode codons that end in a purine (R) (i.e. NNR), tRNAs frequently utilize 5-methyluridine derivatives (xm(5)U) at the wobble position. However, the functional properties of the C5-substituents of xm(5)U in codon recognition remain elusive. We previously found that mitochondrial tRNAs(Leu(UUR)) with pathogenic point mutations isolated from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) patients lacked the 5-taurinomethyluridine (taum(5)U) modification and caused a decoding defect. Here, we constructed Escherichia coli tRNAs(Leu(UUR)) with or without xm(5)U modifications at the wobble position and measured their decoding activities in an in vitro translation as well as by A-site tRNA binding. In addition, the decoding properties of tRNA(Arg) lacking mnm(5)U modification in a knock-out strain of the modifying enzyme (DeltamnmE) were examined by pulse labeling using reporter constructs with consecutive AGR codons. Our results demonstrate that the xm(5)U modification plays a critical role in decoding NNG codons by stabilizing U.G pairing at the wobble position. Crystal structures of an anticodon stem-loop containing taum(5)U interacting with a UUA or UUG codon at the ribosomal A-site revealed that the taum(5)U.G base pair does not have classical U.G wobble geometry. These structures provide help to explain how the taum(5)U modification enables efficient decoding of UUG codons.

Estimation of single-nucleotide polymorphism allele frequency by alternately binding probe competitive polymerase chain reaction.

Estimation of single-nucleotide polymorphism (SNP) allele frequency in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present a simple, accurate, and cost-effective method for estimating SNP allele frequency, called alternately binding probe (ABProbe) competitive polymerase chain reaction (ABC-PCR) that entails no expensive devices for real-time fluorescence measurement and complex post-PCR steps. We prepared DNA pools of PCR products derived from homozygous samples of three different SNPs (ALDH2, GNB3, and HTR2A) in different portions, and the allele frequencies of these samples were estimated by ABC-PCR. Two alleles were coamplified by PCR with a fluorescent probe that binds to either alleles, and then fluorescence intensity was measured using a simple fluorometer. The ratio of the two alleles was calculated from the fluorescence intensity of the probe at the end-point. The estimated allele frequencies strongly correlated to the expected ratios for all three SNPs with high accuracy. When the allele frequencies were more than 5%, the relative standard deviations (R.S.D.s) of ABC-PCR were less than 20%. Moreover, we also confirmed that this method was applicable to SNP genotyping.

Retrieval of entire genes from environmental DNA by inverse PCR with pre-amplification of target genes using primers containing locked nucleic acids.

We had been unsuccessful to amplify desired nucleotide sequences from various environmental DNA samples by using the inverse polymerase chain reaction (IPCR) technique, most probably because the copy numbers of target DNA sequences had been quite low. To enrich the target DNA sequences prior to IPCR, a rolling-circle amplification was used with a site-specific primer containing locked nucleic acids (LNAs). This pre-amplified IPCR (PAI-PCR) method increased the sensitivity of PCR almost 10,000 times compared with the standard IPCR in model experiments using Escherichia coli. We then applied the PAI-PCR method to isolate glycosyl hydrolase genes from DNAs extracted from vermiform appendixes of horses and termite guts. The flanking sequences of the target genes were amplified and cloned successfully using PAI-PCR, whereas standard IPCR resulted in no amplification.

Calibration-curve-free quantitative PCR: a quantitative method for specific nucleic acid sequences without using calibration curves.

We have developed a simple quantitative method for specific nucleic acid sequences without using calibration curves. This method is based on the combined use of competitive polymerase chain reaction (PCR) and fluorescence quenching. We amplified a gene of interest (target) from DNA samples and an internal standard (competitor) with a sequence-specific fluorescent probe using PCR and measured the fluorescence intensities before and after PCR. The fluorescence of the probe is quenched on hybridization with the target by guanine bases, whereas the fluorescence is not quenched on hybridization with the competitor. Therefore, quench rate (i.e., fluorescence intensity after PCR divided by fluorescence intensity before PCR) is always proportional to the ratio of the target to the competitor. Consequently, we can calculate the ratio from quench rate without using a calibration curve and then calculate the initial copy number of the target from the ratio and the initial copy number of the competitor. We successfully quantified the copy number of a recombinant DNA of genetically modified (GM) soybean and estimated the GM soybean contents. This method will be particularly useful for rapid field tests of the specific gene contamination in samples.

Technique for quantitative detection of specific DNA sequences using alternately binding quenching probe competitive assay combined with loop-mediated isothermal amplification.

We describe a novel technique for a simple, rapid, and reliable quantitative detection of specific DNA sequences using an alternately binding quenching probe (AB-QProbe) that binds to either the gene of interest (target) or an internal standard (competitor) in combination with loop-mediated isothermal amplification (LAMP). The AB-QProbe is a singly labeled oligonucleotide bearing a fluorescent dye at the 5' end. The fluorescence intensity of the AB-QProbe reflects the ratio of the LAMP products from the target and competitor. We amplified the target and competitor by LAMP under isothermal conditions with high specificity, efficiency, and rapidity and calculated the starting quantity of the target from the fluorescence intensities at the beginning and end of LAMP. We call this technique alternately binding quenching probe competitive LAMP (ABC-LAMP). We quantified amoA, which encodes the ammonia-oxidizing enzyme in environmental bacteria, as a model target by ABC-LAMP, real-time PCR, and real-time turbidimetry of LAMP. By comparison, the accuracy of ABC-LAMP was found to be similar to that of real-time PCR. Moreover, ABC-LAMP enables the accurate quantification of DNA in the presence of DNA amplification inhibitors such as humic acid, urea, and Triton X-100 that compromise the values measured by real-time PCR and real-time turbidimetry of LAMP.

Quantitative method for specific nucleic acid sequences using competitive polymerase chain reaction with an alternately binding probe.

We have developed a simple, cost-effective, and accurate method for the quantification of specific nucleic acid sequences by the combined use of competitive PCR and a sequence-specific fluorescent probe that binds to either the gene of interest (target) or internal standard (competitor), referred to as alternately binding probe (ABProbe). In this method, the target and competitor were coamplified with the ABProbe, and then the fluorescence intensity was measured. The ratio of the target to the competitor can be calculated from the fluorescence intensity of the ABProbe using fluorescence quenching and fluorescence resonance energy transfer, that is, the starting quantity of the target is successfully calculated by end-point fluorescence measurement. Therefore, this method eliminates the complex post-PCR steps and expensive devices for real-time fluorescence measurement. We called this method alternately binding probe competitive PCR (ABC-PCR). We quantified amoA as a model target by ABC-PCR and real-time PCR. By comparison, the sensitivity, accuracy, and precision of ABC-PCR were similar to those of real-time PCR. Moreover, ABC-PCR was able to correctly quantify DNA even when PCR was inhibited by humic acid; therefore, this method will enable accurate DNA quantification for biological samples that contain PCR inhibitors.

Characterization and quantification of ammonia-oxidizing bacteria in eutrophic coastal marine sediments using polyphasic molecular approaches and immunofluorescence staining.

Tokyo Bay, a eutrophic bay in Japan, receives nutrients from wastewater plants and other urban diffuse sources via river input. A transect was conducted along a line from the Arakawa River into Tokyo Bay to investigate the ecological relationship between the river outflow and the distribution, abundance and population structure of ammonia-oxidizing bacteria (AOB). Five surficial marine sediments were collected and analysed with polyphasic approaches. Heterogeneity and genetic diversity of beta-AOB populations were examined using restriction fragment length polymorphism (RFLP) analysis of 16S rRNA and amoA genes. A shift of the microbial community was detected in samples along the transect. Both 16S rRNA and amoA genes generated polymorphisms in the restriction profiles that were distinguishable at each sampling site. Two 16S rRNA gene libraries were constructed using the reverse transcription polymerase chain reaction (RT-PCR) method to determine the major ammonia oxidizers maintaining high cellular rRNA content. Two major groups were observed in the Nitrosomonas lineage; no Nitrosospira were detected. The effort to isolate novel AOB was successful; the isolate dominated in the gene libraries. For quantitative analysis, a real-time PCR assay targeting the 16S rRNA gene was developed. The population sizes of beta-AOB ranged from 1.6 x 10(7) to 3.0 x 10(8) cells g(-1) in dry sediments, which corresponded to 0.1-1.1% of the total bacterial population. An immunofluorescence staining using anti-hydroxylamine oxidoreductase (HAO) antibody was also tested to obtain complementary data. The population sizes of ammonia oxidizers ranged between 2.4 x 10(8) and 1.2 x 10(9) cells g(-1) of dry sediments, which corresponded to 1.2-4.3% of the total bacterial fraction. Ammonia-oxidizing bacteria cell numbers deduced by the two methods were correlated (R = 0.79, P < 0.01). In both methods, the number of AOB increased with the distance from the river mouth; ammonia-oxidizing bacteria were most numerous at B30, where the ammonium concentration in the porewater was markedly lower and the nitrite concentration was slightly higher than nearby sites. These results reveal spatial distribution and shifts in the population structure of AOB corresponding to nutrients and organic inputs from the river run-off and phytoplankton bloom.

Quantification of genetically modified soybean by quenching probe polymerase chain reaction.

Quenching probe (QProbe) polymerase chain reaction (PCR) is a simple and cost-effective real-time PCR assay in comparison with other real-time PCR assays such as the TaqMan assay. We used QProbe-PCR to quantify genetically modified (GM) soybean (Roundup Ready soybean). We designed event-specific QProbes for Le1 (soy endogenous gene) and RRS (recombinant gene), and we quantified certified reference materials containing 0.1, 0.5, 1, 2, and 5% GM soybean. The TaqMan assay was also applied to the same samples, and the results were compared. The accuracy of QProbe-PCR was similar to that of TaqMan assay. When GM soybean content was 0.5% or more, the relative standard deviations of QProbe-PCR were less than 20%. QProbe-PCR is sensitive enough to monitor labeling systems and has acceptable levels of accuracy and precision.

Reevaluation and reduction of a PCR bias caused by reannealing of templates.

We reevaluated the bias toward a 1:1 ratio of products in multitemplate PCR used in ecological studies and showed that the template reannealing at the annealing step would not cause the bias; however, the preferential homoduplex formation during temperature decrease from denaturation to annealing step would cause the bias.

A FRET-based analysis of SNPs without fluorescent probes.

Fluorescence resonance energy transfer (FRET) is a simple procedure for detecting specific DNA sequences, and is therefore used in many fields. However, the cost is relatively high, because FRET-based methods usually require fluorescent probes. We have designed a cost-effective way of using FRET, and developed a novel approach for the genotyping of single nucleotide polymorphisms (SNPs) and allele frequency estimation. The key feature of this method is that it uses a DNA-binding fluorogenic molecule, SYBR Green I, as an energy donor for FRET. In this method, single base extension is performed with dideoxynucleotides labeled with an orange dye and a red dye in the presence of SYBR Green I. The dyes incorporated into the extended products accept energy from SYBR Green I and emit fluorescence. We have validated the method with ten SNPs, which were successfully discriminated by end-point measurements of orange and red fluorescence intensity in a microplate fluorescence reader. Using a mixture of homozygous samples, we also confirmed the potential of this method for estimation of allele frequency. Application of this strategy to large-scale studies will reduce the time and cost of genotyping a vast number of SNPs.

A new approach to SNP genotyping with fluorescently labeled mononucleotides.

Fluorescence resonance energy transfer (FRET) is one of the most powerful and promising tools for single nucleotide polymorphism (SNP) genotyping. However, the present methods using FRET require expensive reagents such as fluorescently labeled oligonucleotides. Here, we describe a novel and cost-effective method for SNP genotyping using FRET. The technique is based on allele-specific primer extension using mononucleotides labeled with a green dye and a red dye. When the target DNA contains the sequence complementary to the primer, extension of the primer incorporates the green and red dye-labeled nucleotides into the strand, and red fluorescence is emitted by FRET. In contrast, when the 3' end nucleotide of the primer is not complementary to the target DNA, there is no extension of the primer, or FRET signal. Therefore, discrimination among genotypes is achieved by measuring the intensity of red fluorescence after the extension reaction. We have validated this method with 11 SNPs, which were successfully determined by end-point measurements of fluorescence intensity. The new strategy is simple and cost-effective, because all steps of the preparation consist of simple additions of solutions and incubation, and the dye-labeled mononucleotides are applicable to all SNP analyses. This method will be suitable for large-scale genotyping.

Quick two-step RNA ligation employing periodate oxidation.

The introduction of modified or labeled nucleotides into RNA is a powerful RNA engineering tool as it enables us to investigate how native RNA modifications affect RNA function and structure. It also helps in the structural analysis of RNA. A modified nucleotide can be introduced into a specific position of RNA by the method of two-step enzymatic ligation of RNA fragments. However, this method requires a complicated purification step between the two ligation steps that results in low yields of the ligation product. Here we have developed a new ligation technique employing periodate oxide that eliminates this purification step. This increases the total yield of the ligation product and makes it a faster procedure.

Decoding property of C5 uridine modification at the wobble position of tRNA anticodon.

Post-transcriptional modification at the first (wobble) position of the tRNA anticodon participates in precise decoding of the genetic code. We recently identified a novel taurine-containing modified uridine (tau m5U; 5-taurinomethyluridine) at the wobble position of mammalian mitochondrial tRNAs and found lack of this modification in mutant mitochondrial tRNAs from human pathogenic cells of the mitochondrial encephalomyopathies, investigate molecular pathogenesis of the diseases, decoding activity of wobble uridines with or without C5 modification was measured using E. coli cell-free translation system. It has been revealed that C5 modification has a functional role for stabilizing U:G wobble base pair.