A universal method for automated gene mapping

Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost.     

A modified universal fast walking method for single-tube transposon mapping

An enhanced universal fast walking (UFW) method adapted for the mapping of transposons is described. This protocol combines the original UFW method with the use of agarase to unravel composite nucleotide sequence, thereby forgoing molecular cloning steps and the use of restriction enzymes and ligases necessary in other available genome walking methods such as the prominent inverse PCR. The minuscule automatable chemistry of UFW is completed within one reaction vessel using a constant enzyme buffer, and the intrinsic DNA fingerprints, from which amplicons may be quantitatively recovered, offer quality assurance. The core steps of the protocol, spanning half a day or less, comprise first-strand synthesis, primer destruction, random-ended-primer annealing, distal branched-end repair, second-primer destruction, lariat formation and final amplification. Distinctively, no starting or intermediate templates are wasted during the reaction series, thus achieving yields comparable to direct PCR. Ultimate per-reaction walk-lengths are schematically illimitable and sequence-ready amplicons can be produced immediately from prevalent single-copy genomic walk origins. The core UFW protocol may be applied, as described here, to expedited transposon boundary retrieval, but is also applicable to general genome walking and cDNA walking, as well as viral and other insertional element mapping.     

A universal airborne LiDAR approach for tropical forest carbon mapping

Airborne light detection and ranging (LiDAR) is fast turning the corner from demonstration technology to a key tool for assessing carbon stocks in tropical forests. With its ability to penetrate tropical forest canopies and detect three-dimensional forest structure, LiDAR may prove to be a major component of international strategies to measure and account for carbon emissions from and uptake by tropical forests. To date, however, basic ecological information such as height–diameter allometry and stand-level wood density have not been mechanistically incorporated into methods for mapping forest carbon at regional and global scales. A better incorporation of these structural patterns in forests may reduce the considerable time needed to calibrate airborne data with ground-based forest inventory plots, which presently necessitate exhaustive measurements of tree diameters and heights, as well as tree identifications for wood density estimation. Here, we develop a new approach that can facilitate rapid LiDAR calibration with minimal field data. Throughout four tropical regions (Panama, Peru, Madagascar, and Hawaii), we were able to predict aboveground carbon density estimated in field inventory plots using a single universal LiDAR model (r ²  = 0.80, RMSE = 27.6 Mg C ha⁻¹). This model is comparable in predictive power to locally calibrated models, but relies on limited inputs of basal area and wood density information for a given region, rather than on traditional plot inventories. With this approach, we propose to radically decrease the time required to calibrate airborne LiDAR data and thus increase the output of high-resolution carbon maps, supporting tropical forest conservation and climate mitigation policy.     

A simple,universal, efficient PCR-based gene synthesis method: Sequential OE-PCR gene synthesis

Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience.     

DNAWorks: an automated method for designing oligonucleotides for PCR-based gene synthesis

The availability of sequences of entire genomes has dramatically increased the number of protein targets, many of which will need to be overexpressed in cells other than the original source of DNA. Gene synthesis often provides a fast and economically efficient approach. The synthetic gene can be optimized for expression and constructed for easy mutational manipulation without regard to the parent genome. Yet design and construction of synthetic genes, especially those coding for large proteins, can be a slow, difficult and confusing process. We have written a computer program that automates the design of oligonucleotides for gene synthesis. Our program requires simple input information, i.e. amino acid sequence of the target protein and melting temperature (needed for the gene assembly) of synthetic oligonucleotides. The program outputs a series of oligonucleotide sequences with codons optimized for expression in an organism of choice. Those oligonucleotides are characterized by highly homogeneous melting temperatures and a minimized tendency for hairpin formation. With the help of this program and a two-step PCR method, we have successfully constructed numerous synthetic genes, ranging from 139 to 1042 bp. The approach presented here simplifies the production of proteins from a wide variety of organisms for genomics-based studies.     

A universal method for detection of amyloidogenic misfolded proteins

Diseases associated with the misfolding of endogenous proteins, such as Alzheimer's disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases.     

A simple method for mapping aquatic macrophytes

Aerial photography with a balloon-suspended camera is a suitable tool for surveying aquatic vegetation and for measuring water movements. Examples from the lake Lunzer Untersee (Austria) are given.     

A sensitive automated method for adenosine triphosphatase kinetics.

An automated method for measuring adenosine triphosphatase (ATPase) activity is described. A modified version of a Technicon Autoanalyzer utilizing a sensitive colourimetric technique for determining inorganic phosphate concentrations (1 nmol/ml) allows investigations on enzymes of low specific activities. Dialysis may be used for measuring tissue homogenate activities or bypassed by examining purified enzyme preparations. When combined with a gradient apparatus, the proposed technique is particularly well suited for the study of enzyme kinetics in relation to cation or anion concentrations.     

A novel and universal method for microRNA RT-qPCR data normalization

Gene expression analysis of microRNA molecules is becoming increasingly important. In this study we assess the use of the mean expression value of all expressed microRNAs in a given sample as a normalization factor for microRNA real-time quantitative PCR data and compare its performance to the currently adopted approach. We demonstrate that the mean expression value outperforms the current normalization strategy in terms of better reduction of technical variation and more accurate appreciation of biological changes.     

WebFARM: web server for finite automated restriction mapping

Restriction endonucleases are indispensable tools in molecular biology and biotechnology. Type II restriction endonucleases are part of restriction modification systems. DNA fragment extraction and restriction mapping are the basis for several biotechnological activities. WebFARM is a server application for identifying restriction endonuclease recognition sites and to give information regarding restriction mapping for given nucleotide sequences. WebFARM analyses given nucleotide sequence and identify restriction site for selected restriction endonucleases. It will also provide frequency of restriction for each restriction endonuclease. AVAILABILITY: http://webfarm.bioinfoindia.org/     

A method for the rapid automated assessment of olfactory function

We have developed a strategy for the rapid high-throughput screening of odor responsivity in genetically altered mice (in fact, any experimentally altered animal). Specifically, the report presents the development and validation of a fully automated procedure based on the evaluation of an animal's stimulus-induced reflexive breathing response (i.e. sniffing behavior) to both air and odorant stimuli. The method requires no training of the animal to be screened and the outcome of the evaluation yields an operationally defined measure. Briefly, using whole-body plethysmography, the procedure determines the numerical values for a set of 14 respiratory measures in response to the presentation of air and a well-above-threshold concentration of the odorant propanol. These measures of stimulus-induced sniffing are incorporated into a model that defines a single univariate measure of response behavior, or 'Sniffing Index', for each screened animal. The approach significantly discriminated between the reflexive sniffing response of a control group of mice and that of an experimentally defined manipulated group for which, a priori, we expected to observe a robust altered breathing response to odorant stimulation (i.e. non-odor-aversion-conditioned versus odor-aversion-conditioned C57BL/6J mice). Further, the procedure was able to significantly discriminate between a mutant phenotype with documented alterations in physiologic and behavioral function (namely, the OMP-null mutant), and their background strain. In addition, applying epidemiologic screening principles to the observed data, we established an operational procedure for the evaluation of unknown animals.     

A convenient automated method for the determination of proteolytic enzymes

An automated method for the assay of proteolytic enzymes is described. The insoluble dye-protein complex, Remazolbrilliant Blue-hide powder, is used as the substrate and is readily digested by trypsin, elastase, subtilisin, thermolysin, chymotrypsin, and to a lesser extent pronase, pepsin, and bacterial collagenase. The proteolytic activity of crude microbial culture preparations is expressed in terms of an equivalent concentration of crystalline trypsin, which itself can be readily determined within the concentration range 0.25–5.0 μg/ml.     

一种简单通用的鸟类性别分子鉴定技术（简报）

我国幅员辽阔,生物类型多种多样,是世界上拥有鸟类种类最多的国家之一。截至1999年底,已知有鸟类1253种948亚种,隶属于21目83科,其中有多种为我国特有或珍稀濒危的鸟类。近年来由于环境污染、植被破坏及非法捕猎等种种原因,许多鸟类尤其是珍稀鸟类正逐步濒临灭绝。为了加紧     

Towards universal systems for recombinant gene expression

Recombinant gene expression is among the most important techniques used both in molecular and medical research and in industrial settings. Today, two recombinant expression systems are particularly well represented in the literature reporting on recombinant expression of specific genes. According to searches in the PubMed citation database, during the last 15 years 80% of all recombinant genes reported on in the literature were expressed in either the enterobacterium Escherichia coli or the methylotropic yeast Pichia pastoris. Nevertheless, some eukaryotic proteins are misfolded or inadequately posttranslationally modified in these expression systems. This situation demands identification of other recombinant expression systems that enable the proper expression of the remaining eukaryotic genes. As of now, a single universal system allowing expression of all target genes is still a distant goal. In this light, thorough experimental screening for systems that can yield satisfying quantity and quality of target protein is required. In recent years, a number of new expression systems have been described and used for protein production. Two systems, namely Drosophila melanogaster S2 insect cells and human embryonic kidney 293 (HEK293) cells stably expressing the EBNA-1 gene, show exceptional promise. The time has come to identify a few well-performing systems that will allow us to express, purify, and characterize entire eukaryotic genomes.     

An automated method for acetylcholinesterase kinetics

An automated assay for acetylcholinesterase (EC 3.1.1.7.) has been developed based on the manual spectrophotometric method of Ellmanet al. (1). This method was used to determine (a) the enzyme activity of an unknown sample and (b) the dependence of initial rates given by a fixed enzyme concentration on the substrate concentration. Methods to minimize possible enzyme modification by DTNB (2) are described. Finally a modification of the conventional autoanalyser procedure permitted rapid and reproducible enzyme kinetic analysis under various conditions. This helped to minimize the effects of possible enzyme inactivation at high dilutions especially when using crude enzyme preparations.     

A ratiometric imaging method for mapping ion flux densities

A heterogeneous distribution of ion channels on the cell surface is a prerequisite for several cellular functions. Thus, there has been considerable interest in methods allowing the mapping of ion channel distributions. Here we report on a novel ratiometric imaging technique appropriate to measure spatially resolved ion flux signals by using ion sensitive dyes. However, given that certain relevant cell properties like the surface to volume ratio may exhibit significant spatial heterogeneities, the local influx signal cannot be interpreted as a measure of the local open channel concentration or flux density. To overcome this problem, we suggest an internal normalization procedure, which, in analogy to, but clearly distinct from, well-established ratioing techniques, eliminates effects which would otherwise obscure the desired result. Ratioing is performed on flux signals from a given cell, triggered by two different, subsequent stimuli. If the two stimuli address different ion channels, the flux density distribution caused by two channel types can be determined relative to each other. In cases where one of the stimuli triggers a spatially homogeneous flux signal, ratioing yields an ion flux density map for a given channel type. Thus distribution patterns of ion channels active during a given stimulus may be derived.