Abstract
Backgroundthe use of specific but partially degenerate primers for nucleic acid hybridisations and PCRs amplification of known or unknown gene families was first reported well over a decade ago and the technique has been used widely since then.Resultshere we report a novel and successful selection strategy for the design of hybrid partially degenerate primers for use with RT-PCR and RACE-PCR for the identification of unknown gene families. The technique (named PaBaLiS) has proven very effective as it allowed us to identify and clone a large group of mRNAs encoding neurotoxin-like polypeptide pools from the venom of Agelena orientalis species of spider. Our approach differs radically from the generally accepted CODEHOP principle first reported in 1998. Most importantly, our method has proven very efficient by performing better than an independently generated high throughput EST cloning programme. Our method yielded nearly 130 non-identical sequences from Agelena orientalis, whilst the EST cloning technique yielded only 48 non-identical sequences from 2100 clones obtained from the same Agelena material. In addition to the primer design approach reported here, which is almost universally applicable to any PCR cloning application, our results also indicate that venom of Agelena orientalis spider contains a much larger family of related toxin-like sequences than previously thought.Conclusionwith upwards of 100,000 species of spider thought to exist, and a propensity for producing diverse peptide pools, many more peptides of pharmacological importance await discovery. We envisage that some of these peptides and their recombinant derivatives will provide a new range of tools for neuroscience research and could also facilitate the development of a new generation of analgesic drugs and insecticides.
Highlights
Toxins and toxin-like molecules are present and used widely throughout the animal kingdom
Our approach to the problem of cloning a potentially large peptide family of toxin-like peptides consisted of a number of logical steps, key enabling assumptions and technical solutions which eventually led to the identification of over 100 distinct cDNAs, and which can be applied to any similar problem of identifying large families of expressed peptides or proteins
The RACE reverse transcriptase polymerase chain reaction (RT-PCR) strategy is a useful technique for amplifying specific regions of mRNA between a defined and known internal site sequence and an unknown sequence located at either the 3' or 5' end
Summary
Toxins and toxin-like molecules are present and used widely throughout the animal kingdom. Among the arthropods, which constitute over 90% of the animal kingdom and include bees, wasps, ants, spiders, scorpions and many other various taxa, many are well known for their predacity and toxic venoms. Spider toxins are thought to have derived from a small number of gene super-families with many peptide toxins sharing structural features, conserved amino acids and consensus sequences. This allows them to interact with specific targets such as related classes of cellular receptors. The resultant pool of genes has subsequently been subjected to the pressures of adaptive evolution and this has culminated in the vast arrays of species-specific combinatorial peptide libraries which exist [4]. It is by the very nature of these processes that an opportunity to discover new peptides of pharmacological importance has become possible using techniques such as PCR-based cloning incorporating degenerate primers, and EST-based cloning
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