Gene enriched cDNA library

The peppermint oil gland secretory cell cDNA library has proven to provide a highly enriched source of candidate genes involved in essential oil biosynthesis. A functional genomics approach has successfully been employed to clone genes involved in the mevalonate-independent pathway of isoprenoid biosynthesis and in the peppermint-specific steps producing (-)-menthol and (-)-menthone. The optimization of LC-MS technology to profile phosphoiylated carbohydrates and... 

While cDNA libraries can be used to obtain many genes in a short time frame, it may not yield genes of toxicologic importance. To enrich for these genes there are several methods that rely on obtaining mRNAs that are differentially expressed after treatment. These methods, which are described below, include subtractive hybridizations, DD RT-PCR and SAGE. 

A preproricin gene from Ricinus communis has been characterized by both cDNA [31] and genomic [150] cloning. Southern blot analysis has indicated that the Ricinus communis genome includes at least six lectin genes [150], In our laboratory we constructed a cDNA library using ripening castor bean endosperm poly(A)+ RNA which had been enriched for lectin precursor... 

The cleanup step with streptavidin beads serves to remove the first strand cDNA. What remains is a population of single-stranded molecules that is highly enriched for target of interest. Removal of other, unrelated cDNAs eliminates the imbalance that exists due to the fact that all molecules can anneal to one primer (the library-specific one), but only a tiny fraction anneals to the gene-specific primer. Reduction of this imbalance tremendously improves subsequent PGR performance. 

Importantly, these genomic libraries can be enriched for the appropriate gene transcripts if the tissue or cell type from which the genetic material is harvested conesponds to the site of natural product production. For example, 25% of the clones in cDNA obtained exclusively from the oil gland secretory cell of peppermint Mentha x piperita) appear to be involved in oil metabolism [28]. In another example, mRNA for construction of a C. roseus cDNA Ubraiy obtained from epidermal cells resulted in a collection enriched in genes involved in alkaloid biosynthesis [21]. 

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