A new technique of PCR hot start using oligonucleotide primers with a stem-loop structure is developed here. The molecular beacon oligonucleotide structure without any chromophore addition to the ends was used. The 3'-end sequence of the primers was complementary to the target and five or six nucleotides complementary to the 3'-end were added to the 5'-end. During preparation of the reaction mixture and initial heating, the oligonucleotide has a stem-loop structure and cannot serve as an effective primer for DNA polymerase. After heating to the annealing temperature it acquires a linear structure and primer extension can begin.
Virulence of C. albicans strains can be tested using a mouse model of haematogenously disseminated Candida cells. Initial steps of host-pathogen contact such as adhesion and colonization are not taken into account due to the injection of Candida cells into the blood stream. Here we describe an assay, based on the ex vivo usage of porcine intestinal epithelium (PIE), that is useful to monitor the early stages of a C. albicans infection. The ability of C. albicans to undergo morphogenetic switching between yeast and hyphal stages is thought to contribute to its virulence. We found that hyphal formation was required to allow cells to colonize the PIE. The non-filamentous mutant strains efg1/cph1 which lacks two of the central transcription factors that are required to promote hyphal growth and wal1 that carries a deletion of the C. albicans homolog of the human Wiskott-Aldrich Syndrome Protein and is deficient in endocytosis showed only weak adherence. Furthermore, the wal1 mutant was found to be reduced in virulence using the mouse tail vein injection assay. We also analyzed the colonization properties of a variety of other mutant strains carrying deletions of either secreted aspartyl proteinase (SAP)-family genes or amino acid permease encoding genes (GAP1, SSY1, and PUT4). Interestingly, the nag5 strain which lacks an N-acetylglucosamine kinase showed enhanced filamentation and invasive growth as well as increased resistance against farnesol.
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