Monday, October 10, 2011

Messenger RNA

A great many genes are transcribed constitutively by RNA polymerase II 13 , and it is clear that large quantities of heterogeneous nuclear RNA (hnRNA) are turned over in the nucleus.In eukaryotic cells, messenger RNAs (mRNA) are derived from precursor hnRNA through a series of modifying reactions, which include formation of the 5 cap, methylation, splicing, 3 end processing, and frequently, polyadenylation. Only 1 – 3% of the total RNA in the cytoplasm of a typical eukaryotic cell is mature mRNA. RNA is produced at different rates from different loci; therefore, each mRNA species is classified based on its cytoplasmic prevalence or, more properly, its abundance. There are three official such categories, high abundance, medium abundance, and low abundance mRNAs and, in the mind of this Author, the unofficial very low abundance category.
Highly abundant transcripts are present in hundreds of copies per cell. These are most often observed when a cell is producing an enormous quantity of a particular protein or is high specialized or differentiated to perform a unique function. Medium abundance transcripts are best thought of as being present in dozens of copies per cell; many genes with housekeeping 14 functions manifest their mRNAs at this level of prevalence in the cell. Low abundance mRNAs are generally prevalent in 10 or fewer copies per cell and often are difficult to assay
by many of the older classical techniques, such as Northern analysis , without some form of enrichment in order to increase the statistical probability that such rare messages will be detectable. Very low bundance mRNAs are those present in fewer than one copy per cell, a designation which generally is generally associated with heterogeneous tissue samples or, very commonly, in cases where cancer cells growin in culture manifest a variable, heterogeneous karyotype. In the past these types of mRNAs were referred to as the “ hard to clone genes ” , though newer methods.
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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.