Identification Of The LongSAGE Tags Of Candida. Albicans With GLGI Technique

Candida albicans is an important pathogen, causing the majority of fungal infections in humans. These can range from relatively minor surface infections, such as thrush and vaginal yeast infections, to more serious and life-threatening systemic infections, particularly in immunodeficiency individuals.Candida albicans expresses several virulence factors that contribute to pathogenesis. These factors include host recognition biomolecules (adhesins), secreted aspartyl proteases and phospholipases, morphogenesis (the reversible transition between unicellular yeast cells and filamentous, growth forms). Additionally,'phenotypic switching'is accompanied by changes in antigen expression.The genes expressed in yeast and hyphal phase induced from C.albicans have been analyzed qualitatively and quantitatively with long serial analysis of gene expression (LongSAGE) protocol in our laboratry,and then the genes expression profiles were established.It is a baisis to find out the relationship of the expressed genes in yeast and hyphal cells to phase transition in C.albicans by the genes functional clustering analysis.We have used a technique called the generation of longer cDNA fragments from serial analysis of gene expression (SAGE) tags for gene identification (GLGI), to convert SAGE tags of 17 bases into their corresponding 3'cDNA fragments covering hundred bases. We considered that the amplification of a particular template corresponding to a particular LongSAGE tag would be possible by using a combination of a sense primer containing a LongSAGE tag sequence and an anchored oligo(dT) primer.In this process, only the cDNA templates containing the binding sequences for the SAGE tag will be annealed and extended in the first PCR cycle. In the second cycle, the extension will happen only from the anchored oligo(dT) primer that annealed at the 5'end of the poly(dA) sequences with the anchored nucleotide correctly paired to the last nucleotide before the poly(dA) sequence. Extension of all other anchored primers annealed along the poly(dA) sequences will be blocked because of the presence of the anchor nucleotide. The resulting extended templates...