Typing of Genetic Markers Involved in Stress Response by Fluorescent cDNA-Amplified Fragment Length Polymorphism Technique

Abstract

Identification of genetic markers involved in stress response to physical factors or chemical substances in organisms is a challenging task. Typing of upregulated gene expression due to selective antibacterial pressure is a promising approach in the search of molecular mechanisms responsible for development of resistance. cDNA-Fluorescent Amplified Fragment Length Polymorphism (cDNA-FAFLP) strategy was developed and applied in the search of antimycotic drug resistance marker(s) in medically important fungi as an alternative method to microarray analysis. We compared differential gene expression of two sensitive Candida albicans reference strains (ATCC 10231 and ATCC 60133) and two of their paired resistant to fluconazole and itraconazole mutants. Resistant mutants Candida albicans FLC-R, resistant to fluconazole (MIC > 128 microg/ml) and Candida albicans ICZ-R, resistant to itraconazole (MIC > 4 microg/ml) were obtained in subcultures with gradual increase of the antifungal in the culture medium. cDNA-AFLP profile in both itraconazole resistant mutants showed specific spectrophotometric peaks with 5-6-fold RNA overexpression product of 500 bp length compared to the sensitive strains. Fluconazole mutants do not reveal RNA level changes under tested by us typing conditions. These results indicate that the cDNA-FAFLP strategy is a relatively rapid, simple, and reliable method for simultaneous typing of both constitutive and induced differences in expression of host genes providing insight into the biological processes involved in response to drugs in bacteria and fungi. Moreover, this methodology could be tested for typing of the genome response of any organism to physical or chemical stress factors.

cDNA-AFLP analysis of Psidium guajava L. cultivars under water stress and mechanical injury: methodological implications

“…The cDNA-AFLP provides the expression profile of whole genomes, allowing the monitoring of transcriptional changes related to cellular development and/or environmental modifications (Levterova et al, 2010). The advantage of not requiring previous genome knowledge makes this technique a powerful tool for identifying novel genes in nonmodel organisms.…”

“…The advantage of not requiring previous genome knowledge makes this technique a powerful tool for identifying novel genes in nonmodel organisms. In addition, cDNA-AFLP has high sensitivity and specificity, comparable to microarrays (Levterova et al, 2010).…”

Molecular-Genetic Approaches for Identification and Typing of Pathogenic Candida Yeasts: A Review

“…Fluorescent dye-labelled primers can be used allowing the detection of amplified fragments in gel-based or capillary DNA sequencers. This variant technique is referred to as fluorescent amplified fragment length polymorphism (FAFLP) allowing the highest resolution of all fragments of different size [51], [52]. AFLP usually involves two PCR steps.…”

“…However, AFLP is more reproducible than RAPD as it uses specific primers, and amplification is achieved under high stringency conditions [49], [50]. Although AFLP has proved to be reliable and reproducible as a genotyping method, it has been rarely used for C. albicans typing mainly because it is multiple-step, fairly expensive and requires a relatively high level of expertise [50], [52], [53].…”

Restriction Fragment Length Polymorphism Analysis of PCR-Amplified Fragments (PCR-RFLP) and Gel Electrophoresis - Valuable Tool for Genotyping and Genetic Fingerprinting

“…Applications of cDNA-AFLP include identification of genes associated with sexual maturation (Kang et al, 2011) and identification of genes differentially expressed as a result of the development of antifungal resistance in Candida albicans (Levterova et al, 2010). Recently, cDNA-AFLP was combined with mRNA differential display to produce an improved variant of this technique, robust ordered mRNA differential display (RoDD), for global gene expression profiling (Liu et al, 2011).…”