The first DNA-binding proteins studied by scientists were regu-latory proteins from bacteria, where they act to control perhaps thesimplest genetic systems found in Nature. Many of these bacterialproteins act as ‘repressors’ of gene activity (see the upper part of thepicture) if they bind tightly to a base-sequence of DNAwhich over-laps the ‘promoter’ sequence, where an RNA polymerase enzymecan also bind. They can thereby prevent the binding of RNA poly-merase to a particular promoter, through direct competition for thesame local segment of DNA. In general, such repressor proteinsreduce the rate at which RNAis made from a promoter; and indeedsuch repression of RNA synthesis may be specific to just one or afew genes in an entire organism, if the repressor binds to only oneor a few sites on an entire chromosome.In bacteria, repressor proteins play an important role in reducinglocal rates of transcription; but in plants, animals and other organ-isms whose cells have nuclei – known collectively as eukaryotes1–the chromosome structure itself tends to repress transcription.Indeed, in nucleated organisms it is the activation of genes thatseems to be the more important aspect of gene regulation. Thatprocess is managed by ‘activators’ of transcription that bind specifically to DNAin the general vicinityof a binding site for RNA polymerase. The activator protein maythen increase the rate at which RNA is made, by directly assistingthe RNA polymerase enzyme and its auxiliary proteins to bind at the promoter sequence, through a network of protein-to-protein contacts; or else indirectly by helping to ‘recruit’ enzymes that canchemically modify the chromatin For instance, cer-tain transcription activators may direct histone acetylases to thegeneral region of a specific gene. The resulting modification of his-tones may cause the chromatin to decompact near that promoter,and thereby make it more accessible to RNA polymerase and itsauxiliary proteins.‘DNA looping’ may represent a somewhat more complex exam-ple of how genes are regulated in three dimensions, and not just inone or two, by some linear or planar arrangement of DNA bindingsites. In the latter case, two or more repressor or activator proteinsmay bind to the same piece of DNA, and then join together to cre-ate a small loop or coil, which can affect gene activity very strongly(either positively or negatively) on account of its stable structure.
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.