Although the one mRNA, one polypeptide relationship is widespread among eukaryotes, bicistronic mRNAs have been identified in certain organisms. A bicistronic mRNA is capable of directing the synthesis of two different pro- teins. One might think of biscistronic mRNAs as the eukaryotic answer to the polycistronic mRNAs that are nearly universally observed among prokaryotes. Taking this a step further, functional tricistronic mRNAs, encoding three dif- ferent polypeptides, are in use in certain in vitro applications. Due to the pecu- liarities associated with translation in eukaryotes, the first (upstream) encoded protein is synthesized in the 5 -cap dependent manner usually associated with translation of monocistronic mRNAs while initiation of the synthesis of the second (downstream) polypeptide is under the control by an internal ribos- ome entry site (IRES) which allows ribosome assembly in a non-cap-dependent manner. This translation strategy is widespread among eukaryotic viruses and, while still considered a rarity in higher animal cells, there are reports of bicistronic mRNAs in plants, including the tomato tomPro1 locus and in Arabidopsis (see Farrell and Bassett, 2007 for a recent review). It is also pos- sible for a single-reading-frame mRNA to produce two or more polypeptides by cleavage of large precursor protein (a zymogen, for example), such as with the animal hormones oxytocin and vasopressin (Richter, 1983). These observa- tions have lead investigators to rethink the entire process of the regulation of gene expression and to analyze gene expression data circumspectly.
Tuesday, October 4, 2011
Bioelectronic DNA detection involves forming an electronic circuit mediated by nucleic acid hybridization and it serves as the basis for a DNA detection system called eSensor™ [1-4]. This system uses low-density DNA chips containing electrodes coated with DNA capture probes. Target DNA present in the sample hybridizes specifically both to capture probes and ferrocene labeled signal probes in solution thereby generating an electric current. Currente Sensor DNA chips contain as many as 36 electrodes for simultaneous detection of multiple pathogens from a single sample.
Many pathogens cause both acute and chronic disease at relatively low copy number and may be difficult or impossible to propagate in culture. Thus, most pathogen detection systems rely on nucleic acid amplification by using polymerase chain reaction (PCR). One highly effective amplification strategy targets conserved sequences among the family of organisms of interest. Such broad-range PCR strategies have been used to identify and characterize several known and previously uncharacterized bacteria [5,6] and viruses [7,8]. In order to maximize the utility of these effective pathogen nucleic acid amplification systems, amplification needs to be coupled with rapid, sensitive, and specific detection. Bioelectronic DNA detection by use of the eSensor chip might fulfill this need.
Human papillomaviruses (HPV) serve as an ideal model system for determining the efficiency and feasibility of eSensor DNA detection technology since there are at least 30 distinct genital HPV types that can be effectively amplified with broad-range consensus PCR primers. We designed two eSensor chips, each containing 14 probes specific for the conserved L1 region of the HPV genome. We evaluated clinical cervical cytology samples known to contain one or more HPV types. The eSensor DNA detection platform successfully detected the correct HPV type in most of these clinical samples, demonstrating that the system provides a rapid, sensitive, specific, and economical approach for multiple-pathogen detection and identification from a single sample.Background We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor) in identifying multiple pathogens.