A protocol for high-throughput extraction of DNA from rice leaves

Current DNA isolation methods have limitations between speed and purity in high-throughput molecular genetic analysis such as gene mapping and marker-assisted selection programs. We have optimized a simple and rapid method for isolating high-quality genomic DNA from rice that significantly minimizes time and the use of laboratory materials. One person can process as many as 384 samples in 2 h. The isolated DNA is suitable for polymerase chain reaction-based techniques and is stable for no less than 6 mo of storage at 4°C.     

An improved electroelution method for separation of DNA from humic substances in marine sediment DNA extracts

We present a method for the rapid and simple extraction of DNA from marine sediments using electroelution. It effectively separates DNA from compounds, including humic substances, that interfere with subsequent DNA quantification and amplification. After extraction of the DNA from the sediment into an aqueous solution, the crude sample is encased in 2% agarose gel and exposed to an electrical current, which draws the DNA out of the gel into a centrifugal filter vial. After electroelution, the sample is centrifuged to remove contaminants ≤100 000 Da. Recovery of DNA using this method is quantitative and does not discriminate on the basis of size, as determined using DNA standards and DNA extracts from environmental samples. Amplification of DNA is considerably improved due to removal of PCR inhibitors. For Archaea, only these purified extracts yielded PCR products. This method allows for the use of relatively large volumes of sediment and is particularly useful for sediments containing low biomass such as deeply buried marine sediments. It works with both organic-rich and -poor sediment, as well as with sediment where calcium carbonate is abundant and sediment where it is limited; consequently, adjustment of protocols is unnecessary for samples with very different organic and mineral contents.     

The distribution of dimethylsulfoniopropionate in tropical Pacific coral reef invertebrates

Dimethylsulfoniopropionate (DMSP) is an important component of the global sulfur cycle and may be involved, via its cleavage product dimethylsulfide, in climate regulation. Although it is common in many algae, reports of DMSP in animals, particularly tropical invertebrates, are limited. This study examined the distribution of DMSP in a diverse group of coral reef invertebrates. DMSP was present in all 22 species of cnidarians and ranged from 9 to 723 μmol g⁻¹ of dry mass (DM) with a mean (± 1SD) of 110 ± 180 μmol g⁻¹ DM. It was not detected in a flatworm and an ascidian or in two of five sponges. Concentrations in sponges ranged from undetectable to 16 μmol g⁻¹ DM with a mean of 4 ± 7 μmol g⁻¹ DM. Within the cnidarians, DMSP concentrations did not differ among orders. Among cnidarian species, DMSP concentrations were correlated with symbiotic zooxanthellae densities. Within cnidarian species, DMSP concentrations of individuals were positively correlated with zooxanthellae densities in three of the four species examined. We speculate that DMSP is dietarily derived in sponges and derived from zooxanthellae in the cnidarians. The functions of DMSP in coral reef invertebrates are not known.     

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.     

A high-throughput DNA extraction protocol for tropical molecular breeding programs

Liquid handling robotics and capillary electrophoresis genetic analyzers now offer high-throughput solutions for 2 of the 4 key steps in PCR-based DNA marker-assisted fingerprinting (DNA extraction, PCR amplification, electrophoresis, data analysis). Thus, DNA extraction remains the most significant bottleneck at the bench for large-scale applications in plant breeding and germplasm characterization. We report on a rapid and low-cost method for relatively high-throughput extraction of high-quality DNA from young and mature leaves of sorghum, pearl millet, chickpea, groundnut, and pigeonpea. The procedure uses a modified CTAB/β-mercaptoethanol method for DNA extraction in a 96-well plate. The quantity and quality of the DNA extracted per sample is adequate for more than 1000 PCR reactions. A relatively high throughput of 96–384 samples per person per day can be achieved, depending on the crop. A major timesaving aspect of the protocol is the absence of a manual sample-grinding step. Finally, the cost is a magnitude lower than commercial plate-based kits, and, as such, is likely to have substantial application in tropical molecular breeding programs.     

Independent origins of neurons and synapses: insights from ctenophores

There is more than one way to develop neuronal complexity, and animals frequently use different molecular toolkits to achieve similar functional outcomes. Genomics and metabolomics data from basal metazoans suggest that neural signalling evolved independently in ctenophores and cnidarians/bilaterians. This polygenesis hypothesis explains the lack of pan-neuronal and pan-synaptic genes across metazoans, including remarkable examples of lineage-specific evolution of neurogenic and signalling molecules as well as synaptic components. Sponges and placozoans are two lineages without neural and muscular systems. The possibility of secondary loss of neurons and synapses in the Porifera/Placozoa clades is a highly unlikely and less parsimonious scenario. We conclude that acetylcholine, serotonin, histamine, dopamine, octopamine and gamma-aminobutyric acid (GABA) were recruited as transmitters in the neural systems in cnidarian and bilaterian lineages. By contrast, ctenophores independently evolved numerous secretory peptides, indicating extensive adaptations within the clade and suggesting that early neural systems might be peptidergic. Comparative analysis of glutamate signalling also shows numerous lineage-specific innovations, implying the extensive use of this ubiquitous metabolite and intercellular messenger over the course of convergent and parallel evolution of mechanisms of intercellular communication. Therefore: (i) we view a neuron as a functional character but not a genetic character, and (ii) any given neural system cannot be considered as a single character because it is composed of different cell lineages with distinct genealogies, origins and evolutionary histories. Thus, when reconstructing the evolution of nervous systems, we ought to start with the identification of particular cell lineages by establishing distant neural homologies or examples of convergent evolution. In a corollary of the hypothesis of the independent origins of neurons, our analyses suggest that both electrical and chemical synapses evolved more than once.     

The dried corncob as a source of DNA for PCR analysis

We developed a procedure to isolate DNA from dried corncobs. This DNA was amplified successfully by PCR, producing well-defined bands in response to specific primers. The SSR patterns between cob and leaf DNA of the same inbred line were found to be identical, indicating that the DNA in the cob cells had not degraded during senescence.     

A modified hybridization protocol for low-density diagnostic DNA microarrays

To promote the application of DNa microarrays for clinical diagnosis, the problems of cross-hybridization and low signal intensity in the hybridization processes has been addressed. We tested a new hybridization protocol for low-density diagnostic DNA microarrays, by skipping the purification step during sample labeling, while elevating the hybridization temperature from 42°C to 52°C, adding a step of distilled water rinsing immediately after hybridization and before the low stringency washing steps. It was found that the modified hybridization protocol works well in our study, which increased detection sensitivity and eliminated nonspecific signals.     

A simplified protocol for preparing DNA from filamentous cyanobacteria

The preparation of good quality genomic DNA from microalgae and plants is often time-consuming because of the need to remove contaminants that may interfere with the downstream enzymatic manipulation of the DNA. Simpler protocols have been reported but these are applicable only to a few species and in many cases are not effective for removing trace contaminants. In this report, we describe a modification of existing protocols that significantly simplified the preparation of genomic DNA from cyanobacteria and plants. A key step in our protocol is the precipitation of DNA in a high concentration of salt (2–2.5 M NaCl) in the presence of isopropanol, immediately following phenol and chloroform extractions. The preparation and enzymatic digestion of the DNA can be performed in a single day. The DNA was easily digested in 2 h at normal restriction enzyme concentrations, and is highly suitable for PCR and Southern hybridization. We successfully used this simplified protocol to prepare genomic DNA from several filamentous cyanobacteria, such asAnabaena sp. PCC 7120,Anabaena siamensis, andSpirulina strains M2 and Kenya. This protocol may also be useful for preparing genomic DNA from other algae and from higher plants.     

Purification and characterization of a novel ATP-independent type I DNA topoisomerase from a marine methylotroph

DNA topoisomerase is involved in DNA repair and replication. In this study, a novel ATP-independent 30-kDa type I DNA topoisomerase was purified and characterized from a marine methylotroph, Methylophaga sp. strain 3. The purified enzyme composed of a single polypeptide was active over a broad range of temperature and pH. The enzyme was able to relax only negatively supercoiled DNA. Mg(2+) was required for its relaxation activity, while ATP gave no effect. The enzyme was clearly inhibited by camptothecin, ethidium bromide, and single-stranded DNA, but not by nalidixic acid and etoposide. Interestingly, the purified enzyme showed Mn(2+)-activated endonuclease activity on supercoiled DNA. The N-terminal sequence of the purified enzyme showed no homology with those of other type I enzymes. These results suggest that the purified enzyme is an ATP-independent type I DNA topoisomerase that has, for the first time, been characterized from a marine methylotroph.     

A simple protocol for isolation of fungal DNA

Genomic DNA was isolated from as little as 2 mg dry biomass of Magnaporthe grisea by microwave treatment within 30 s. The quantity of DNA was good enough for PCR analysis and Dot blot hybridization. This technique can be used for various studies, such as DNA fingerprinting to study the population structure of the phytopathogen in different regions, and for a quick screening of M. grisea transformants.     

A modified method for PCR-directed gene synthesis from large number of overlapping oligodeoxyribonucleotides

Here we report an improved, reproducible, simple, rapid, and cost-effective PCR-based DNA synthesis method using short (25–40 bp) overlapping oligodeoxyribonucleotides (oligos). The method involves two steps; (1) assembly of multiple/overlapping oligos by PCR to generate the template DNA and (2) amplification of the template DNA sequence with the two outermost oligos as primers. We have tested this method by synthesizing approximately 35 genes ranging in size between 300 bp and 1700 bp and G + C content from moderate (30%) to high (65%). In addition, we used the method to introduce 29 mutations simultaneously into a single gene. Key to the success of this method is the use of optimized oligo concentrations and the type of DNA polymerase used. This simplified and highly reproducible method is expected to be beneficial for the synthesis of a wide variety of genes.     

The determination of multiple microsatellite genotypes and DNA sequences from a single pollen grain

Pollen plays important roles in the reproduction and gene flow of flowering plants, and its haploid DNA sequence provides useful information for studies of plant evolution and genealogy. We describe a new method for multiple microsatellite genotyping and DNA sequencing from a single pollen grain. The haploid DNA was extracted from a single pollen grain by using a simple DNA extraction method, and multiple microsatellite genotypes and DNA sequences of multiple chloroplast loci were determined. Using nine pairs of microsatellite primers, more than 90% of genotypes were successfully determined, and 71% and 100% of DNA sequences were determined at two chloroplast DNA loci, the trnL intron region and the trnL/trnF intergenic spacer region, respectively. This simple method of genetic analysis for a single pollen grain will facilitate detailed study of pollination, evolution and genealogy.     

用DNA重组法表达丙肝病毒部分NS_4-NS_5基因

用ＤＮＡ重组法表达丙肝病毒部分ＮＳ_４－ＮＳ_５基因祁自柏,谷金莲,李河民（中国药品生物制品检定所,北京１０００５０）丙型肝炎病毒（ＨＣＶ）是一种ＲＮＡ病毒,为研究其复制机理,制备检测丙肝抗体的诊断试剂,需大量纯化的各种丙肝病毒蛋白,我们在国内首次用Ｄ...     

活性污泥总DNA提取方法的研究

采用冻融＋玻璃珠、溶菌酶＋SDS和冻融＋玻璃珠＋溶菌酶＋SDS的方法提取活性污泥的微生物总DNA,并对以上三种方法进行比较,从中确定最佳的方法,以实现普通实验条件下成功提取符合PCR扩增要求的DNA。经紫外光度分析及电泳结果表明,冻融＋玻璃珠＋溶菌酶＋SDS方法所得的DNA的OD260/OD280为1．81,电泳结果显示,所提DNA片段分子量大于10kb,适于酶解和PCR扩增要求,为PCR技术应用于活性污泥的研究提供了一种简便、可靠的DNA提取方法。     

Evaluation of a shortened QIAsymphony DNA extraction protocol for stool samples using a multiplex real-time PCR for the detection of enteric pathogens

A shortened DNA extraction protocol for the QIAsymphony SP was evaluated by quantitative and qualitative comparison of real-time PCR results of 150 co-extracted stool samples. The average ?Cycle threshold value for positive pathogenic targets was 0.28 Ct. A consensus of 96.91%, with a correlation coefficient of 0.9880 was recorded.     

几种耐热DNA聚合酶的比较

耐热DNA聚合酶广泛应用在PCR技术。本文将常用的几种耐热DNA聚合酶分为三大类,对它们的性质、差异、用途做一个简单的介绍。