An overwhelming majority of higher plant and animal (eukaryotic) genes consist of coding regions known as exons that are separated by intervening, non-coding regions known as introns. In the course of gene expression, transcription results in the synthesis of a large, immature pre-mRNA molecules, consisting of both coding (exon) and non-coding (intron) sequences from a specific gene locus. The process of mRNA maturation involves the removal of introns and the joining together (ligation) of exons so that all of the coding information is contiguous. Generically known as splicing, these well-orchestrated events involve the forma- tion of a spliceosome, i.e. an RNA splicing complex, and involves the removal of introns and the ligation of exons from the same RNA molecule. Nearly all of the splicing that occurs in the cell, as described above, is known as cis -splicing because the exons from a single pre-mRNA molecule are ligated together. In contrast, trans -splicing involves the joining of exons from two different RNA molecules, resulting in the formation of a hybrid (chimeric) RNA molecule. Like cis -splicing, trans -splicing is a naturally occurring proc- ess in eukaryotes, albeit at a much, much lower frequency, though it has been reported that as many of 70% of all mRNAs in the nematode C. elegans may be subject to trans- splicing (reviewed by Hastings, 2005 ). Trans - splicing has the potential to be adapted both in vitro and in vivo to produce an astonishing array of designer proteins, not to mention potential to repair defective mRNAs. SMaRT (spliceosome-mediated RNA trans -splicing) technology, a patented spliceosome-mediated trans- splicing process owned by VIRxSYS, attempts to correct cellular damage caused by the formation of aber- rant proteins by fixing or “ reprogramming ” defective pre-mRNA molecules so that only normal proteins are produced, even when a mutation is harbored and persists in the DNA. Naturally occurring transcription produces.
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.