Genes are transcribed by enzymes known as RNA polymerases, thereby pro- ducing the major types of RNA, including ribosomal RNA (rRNA), transfer RNA (tRNA), and mRNA, as well as all of the smaller RNA species. Eukaryotic genes are transcribed by one of four nuclear RNA polymerases; these enzymes are among the largest and most complex proteins in the cell and consist of more subunits than their prokaryotic counterpart. The eukaryotic enzymes are properly known as RNA polymerases I, II, III, and IV, each of which is responsible for transcribing a different class of genes. Prokaryotes, in contrast, exhibit only one type of RNA polymerase, which transcribes all classes of RNA. RNA polymerases are active only in the presence of DNA, and require the nucleotides ATP, CTP, GTP, and UTP as precur- sors, myriad transcription factors. As is the case in the synthesis of all nucleic acid molecules, RNA transcripts are assembled only in the 5 → 3 direction. Transcription involves three distinct phases, namely, initiation, elongation, and termination, all of which have been described in great detail elsewhere and the details of which are beyond the scope of this volume. Briefly, initiation involves the attachment of RNA polymerase to a DNA template promoter, via transcription, activation, and initiation factors, followed by the acquisition of what will be the first ribonu- cleotide in the RNA molecule. Elongation involves the sequential addition of ribonucleotides to the nascent chain, a process also involving accessory protein elongation factors. Termination is the completion of RNA synthesis, whether appropriately or prematurely, and the disengagement of both the newly synthe- sized RNA and the RNA polymerase from the DNA template. Transcription termination, as with initiation and elongation, is sequence-dependent and is influenced by the presence of small proteins (termination factors) as well as the transient formation of RNA 2 ° structures. Mutations notwithstanding, the nucleotide sequence of the resulting RNA molecule is identical to the coding strand of the DNA from which it is derived, the only difference being the sub- stitution of the base uracil for thymine.
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.