The Kirkhouse Trust: Successes and Challenges in Twenty Years of Supporting Independent, Contemporary Grain Legume Breeding Projects in India and African Countries.

This manuscript reviews two decades of projects funded by the Kirkhouse Trust (KT), a charity registered in the UK. KT was established to improve the productivity of legume crops important in African countries and in India. KT's requirements for support are: (1) the research must be conducted by national scientists in their home institution, either a publicly funded agricultural research institute or a university; (2) the projects need to include a molecular biology component, which to date has mostly comprised the use of molecular markers for the selection of one or more target traits in a crop improvement programme; (3) the projects funded are included in consortia, to foster the creation of scientific communities and the sharing of knowledge and breeding resources.

The genesis of microarrays.

This review provides a perspective on the initial development of microarray technologies by two independent groups in the late 1980s.

Reactive trityl derivatives: stabilised carbocation mass-tags for life sciences applications.

The rational design of novel triarylmethyl (trityl)-based mass tags (MT) for mass-spectrometric (MS) applications is described. We propose a "pK(R+) rule" to correlate the stability of trityl carbocations with their MS performance: trityls with higher pK(R+) values ionise and desorb better. Trityl blocks were synthesised that have high pK(R+) values and are stable in conditions of MS analysis; these MTs can be ionised by matrix as well as irradiation with a 337 nm nitrogen laser.

Novel mass tags for single nucleotide polymorphism detection.

A new method suitable for single nucleotide polymorphism detection and other applications based on oligonucleotide probe extension has been developed. The method is based on mass spectrometry and utilizes a single surface for affinity purification of extended probes and matrix-independent desorption/ionization of the cleavable labels. A new family of sulfur-linked laser-cleavable trityl labels with vastly improved flying abilities is implemented in this study.

Multiplex target protein imaging in tissue sections by mass spectrometry--TAMSIM.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) is becoming a popular tool for imaging histological sections. Currently, this technology is used to image naturally occurring molecules. Here we report a novel development for multiplex imaging of candidate proteins.

Electrochemically directed synthesis of oligonucleotides for DNA microarray fabrication.

We demonstrate a new method for making oligonucleotide microarrays by synthesis in situ. The method uses conventional DNA synthesis chemistry with an electrochemical deblocking step. Acid is delivered to specific regions on a glass slide, thus allowing nucleotide addition only at chosen sites.

S(O)-pixyl protecting group as efficient mass-tag.

We report herein the design, preparation and first applications of novel trityl tags with adjustable stability, efficient as protecting groups or MS analytes.

In situ oligonucleotide synthesis on poly(dimethylsiloxane): a flexible substrate for microarray fabrication.

In this paper, we demonstrate in situ synthesis of oligonucleotide probes on poly(dimethylsiloxane) (PDMS) microchannels through use of conventional phosphoramidite chemistry. PDMS polymer was moulded into a series of microchannels using standard soft lithography (micro-moulding), with dimensions <100 microm. The surface of the PDMS was derivatized by exposure to ultraviolet/ozone followed by vapour phase deposition of glycidoxypropyltrimethoxysilane and reaction with poly(ethylene glycol) spacer, resulting in a reactive surface for oligonucleotide coupling.

Structural rearrangements in RNA on the binding of an antisense oligonucleotide: implications for the study of intra-molecular RNA interactions and the design of cooperatively acting antisense reagents with enhanced efficacy.

We show that binding of an antisense oligonucleotide can lead to considerable changes in the target mRNA structure. The approaches described here are not only useful in the study of intra-molecular interactions in RNAs but can also be used to design oligonucleotides that facilitate binding of other antisense reagents. Such "cooperatively acting" antisense reagents have the potential to overcome several problems faced in their use, for example, low efficacy and non-specificity.

A novel anionic dendrimer for improved cellular delivery of antisense oligonucleotides.

The optimal design of hybridisation-competent antisense oligonucleotides (ODNs) coupled with an efficient delivery system appear to be important prerequisites for the successful use of antisense reagents for gene silencing. We selected an antisense ODN complementary to an accessible region of the epidermal growth factor receptor (EGFR) mRNA with the aid of an antisense oligonucleotide scanning array. The scanning array comprised 2684 antisense ODN sequences targeting the first 120 nts in the coding region of EGFR mRNA.

Messenger RNA expression profiling of genes involved in epidermal growth factor receptor signalling in human cancer cells treated with scanning array-designed antisense oligonucleotides.

Scanning oligodeoxynucleotide (ODN) arrays appear promising in vitro tools for the prediction of effective antisense reagents but their usefulness has not yet been reported in mammalian systems. In this study, we have evaluated the use of scanning ODN arrays to predict efficacious antisense ODNs targeting the human epidermal growth factor receptor (EGFR) mRNA in a human epidermoid cancer cell line and in primary human glioma cells. Hybridisation accessibility profile of the first 120nt in the coding region of the human EGFR mRNA was determined by hybridising a radiolabelled EGFR transcript to a scanning array of 2684 antisense sequences ranging from monomers to 27-mers.

A simple and cost-effective method for producing small interfering RNAs with high efficacy.

Small interfering RNAs (siRNAs) are powerful RNA interference (RNAi) reagents for directed post- transcriptional gene silencing. Exogenous siRNA is frequently used in RNAi studies. However, due to profound differences in the activity of siRNAs targeted to different regions of a gene, several reagents may have to be screened for optimal activity.

The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript.

The type 1 insulin-like growth factor receptor (IGF1R) is often overexpressed by tumors and mediates growth and apoptosis protection. We previously showed that antisense reagents complementary to the IGF1R translation start site enhance radio- and chemosensitivity and impair Atm function. However these agents induce relatively modest IGF1R down-regulation and affect insulin receptor levels.

An electrochemical redox couple activitated by microelectrodes for confined chemical patterning of surfaces.

Microelectrodes, printed as an array on the surface of a silicon chip, generate chemically active species in a solution of electrolyte held between the electrode array and a glass plate. The active species induce chemical change in molecules coupled to the surface of the glass plate, which is separated from the electrode array by a gap of several micrometers. This paper explores the nature and pattern of the induced chemical change.

Thermus scotoductus and Rhodothermus marinus DNA ligases have higher ligation efficiencies than thermus thermophilus DNA ligase.

To mimic large numbers of nicked DNA duplexes we used a technique that produces nicked duplex DNA substrates by hybridization of complementary oligonucleotides, adjacent to an initiating primer, which are ligated together by a thermostable DNA ligase. Sequential ligation of nonanucleotides to this primary duplex results in the formation of polymers that can be analyzed by gel electrophoresis. The extent of polymerization is a measure of the efficiency of ligation.