Protocol: Streamline cloning of genes into binary vectors in Agrobacterium via the Gateway® TOPO vector system

<p>Abstract</p> <p>Background</p> <p>In plant functional genomic studies, gene cloning into binary vectors for plant transformation is a routine procedure. Traditionally, gene cloning has relied on restriction enzyme digestion and ligation. In recent years, however, Gateway^®cloning technology (Invitrogen Co.) has developed a fast and reliable alternative cloning methodology which uses a phage recombination strategy. While many Gateway- compatible vectors are available, we frequently encounter problems in which antibiotic resistance genes for bacterial selection are the same between recombinant vectors. Under these conditions, it is difficult, if not sometimes impossible, to use antibiotic resistance in selecting the desired transformants. We have, therefore, developed a practical procedure to solve this problem.</p> <p>Results</p> <p>An integrated protocol for cloning genes of interest from PCR to <it>Agrobacterium </it>transformants via the Gateway^®System was developed. The protocol takes advantage of unique characteristics of the replication origins of plasmids used and eliminates the necessity for restriction enzyme digestion in plasmid selections.</p> <p>Conclusion</p> <p>The protocol presented here is a streamlined procedure for fast and reliable cloning of genes of interest from PCR to <it>Agrobacterium </it>via the Gateway^®System. This protocol overcomes a key problem in which two recombinant vectors carry the same antibiotic selection marker. In addition, the protocol could be adapted for high-throughput applications.</p

Hierarchical Lee-Carter model estimation through data cloning applied to demographically linked countries

Some groups of countries are connected not only economically, but also social and even demographically. This last fact can be exploited when trying to forecast the death rates of their populations. In this paper we propose a hierarchical specification of the Lee-Carter model and we assume that there is a common latent mortality factor for all of them. We introduce an estimation procedure for this kind of structures by means of a data cloning methodology. To our knowledge, this is the first time that this methodology is used in the actuarial field. It allows approximating the maximum likelihood estimates, which are not affected by the prior distributions assumed for the calculus. Finally, we apply the methodology to some France, Italy, Portugal and Spain data. The forecasts obtained using this methodology can be considered as very satisfactory

Molecular cloning using in vivo DNA assembly

Here we describe the in vivo DNA assembly approach, where molecular cloning procedures are performed using an E. coli recA-independent recombination pathway, which assembles linear fragments of DNA with short homologous termini. This pathway is present in all standard laboratory E. coli strains and, by bypassing the need for in vitro DNA assembly, allows simplified molecular cloning to be performed without the plasmid instability issues associated with specialized recombination-cloning bacterial strains. The methodology requires specific primer design and can perform all standard plasmid modifications (insertions, deletions, mutagenesis, and sub-cloning) in a rapid, simple, and cost-efficient manner, as it does not require commercial kits or specialized bacterial strains. Additionally, this approach can be used to perform complex procedures such as multiple modifications to a plasmid, as up to 6 linear fragments can be assembled in vivo by this recombination pathway. Procedures generally require less than 3 h, involving PCR amplification, DpnI digestion of template DNA, and transformation, upon which circular plasmids are assembled. In this chapter we describe the requirements, procedure, and potential pitfalls when using this technique, as well as protocol variations to overcome the most common issues

Improved Screening of cDNAs Generated by mRNA Differential Display Enables the Selection of True Positives and the Isolation of Weakly Expressed Messages

The high percentage of false positives generated by differential display (as high as 85%) has previously limited the potential of the method. This report describes an efficient methodology that enables false positives to be discarded prior to cloning, via reverse Northern analysis. This first step of the screening also allows the detection of putative lowabundance differential clones. Following cloning, a second reverseNorthern combined with partial DNA sequencing and RT-PCR detection allows isolation of all differential cDNAs including very lowabundance clones. Use of the sequential screening procedure described here led to the isolation of novel tomato genes responding to the plant hormone ethylene while minimising labor and materials input

Approximate maximum likelihood estimation using data-cloning ABC

A maximum likelihood methodology for a general class of models is presented, using an approximate Bayesian computation (ABC) approach. The typical target of ABC methods are models with intractable likelihoods, and we combine an ABC-MCMC sampler with so-called "data cloning" for maximum likelihood estimation. Accuracy of ABC methods relies on the use of a small threshold value for comparing simulations from the model and observed data. The proposed methodology shows how to use large threshold values, while the number of data-clones is increased to ease convergence towards an approximate maximum likelihood estimate. We show how to exploit the methodology to reduce the number of iterations of a standard ABC-MCMC algorithm and therefore reduce the computational effort, while obtaining reasonable point estimates. Simulation studies show the good performance of our approach on models with intractable likelihoods such as g-and-k distributions, stochastic differential equations and state-space models.Comment: 25 pages. Minor revision. It includes a parametric bootstrap for the exact MLE for the first example; includes mean bias and RMSE calculations for the third example. Forthcoming in Computational Statistics and Data Analysi

Structured Review of the Evidence for Effects of Code Duplication on Software Quality

This report presents the detailed steps and results of a structured review of code clone literature. The aim of the review is to investigate the evidence for the claim that code duplication has a negative effect on code changeability. This report contains only the details of the review for which there is not enough place to include them in the companion paper published at a conference (Hordijk, Ponisio et al. 2009 - Harmfulness of Code Duplication - A Structured Review of the Evidence)

MRIs and the Perception of Risk

The most important safety decision concerning MRIs was to change the name of the procedure. In the late 1970s, the procedure known as nuclear magnetic resonance (NMR) became magnetic resonance imaging (MRI) because of the negative connotations the word “nuclear” invited. The change was understandable since MRIs do not expose patients to dangerous radiation: “nuclear” was in the original name because basic research on the atomic nucleus led to the development of MRIs. The main cost of the name change was to obscure the important link between basic research and useful medical technologies. In recent years, however, MRIs, a generally safe procedure, have been bothered by a new problem. MRIs, which are used in research as well as medical settings, often result in incidental findings of medical conditions that may be clinically significant. Automatic notification of subjects, however, is not always the sensible route, since incidental findings that turn out to be clinically irrelevant can lead not only to fear and anxiety but to needless and dangerous interventions. Sound ethical policies on incidental findings need to be developed. It is ironic that this process will involve the weighing of patient fears that may not be shared by experts; the original name change was motivated by much the same situation

Oil palm biotechnology: progress and prospects

Today, a range of biotechnological approaches, from somatic embryogenesis to biomolecular research, play an increasingly important role in breeding strategies for oil palm (Elaeis guineensis Jacq.). Clonal micropropagation. Methods of cloning by in vitro culture led to the development of a micropropagation technique for oil palm based on somatic embryogenesis which was tested at the pilot stage on elite genotypes, thus enabling the production of high oil yielding clones. This phase allowed the identification of limiting factors associated with scaling-up, with respect in particular to the scale of mass production required to meet the needs of planters and to the problem of ensuring genetic fidelity in the regenerated plant material. These two concerns led researchers to look further into the underlying physiological and/or molecular mechanisms involved in somatic embryogenesis and the somaclonal variation events induced by the in vitro cloning procedure. Structural and functional genomics. Marker-assisted breeding in oil palm is a long-term multi-stage project including: molecular analysis of genetic diversity in both E. guineensis and E. oleifera germplasms; large scale development of PCR-based microsatellite markers; and parallel development of three genome mapping and QTL detection projects studying key agronomic characters. Post-genomics. In order to tackle the problem of the mantled flowering abnormality, which is induced during the micropropagation process, studies of gene expression have been carried out in tissue cultures as a means of establishing an early clonal conformity testing procedure. It is important to assess what kind of methodology is the most appropriate for clonal conformity testing by comparing RNA, protein and DNA (PCR) based approaches. Parallel studies on genomic DNA methylation changes induced by tissue culture suggest that the latter may play an important role in the determination of the mantled abnormality. (Résumé d'auteur