Sunday, January 1, 2012

dna amplification Result

we designed the amplification linker to ligate to the overhang left by HpyCh4IV digestion and simultaneously to create a site for the relatively rare–cutting enzyme, MluI. When amplification occurs as intended with the linkers ligated to HpyCh4IV sites, subsequent MluI digestion of products cleaves off the linker sequence. Because PCR is performed with a biotinylated primer, products can be bound to streptavidin-coated paramagnetic beads. In cases of illegitimate amplification, the fragment cannot be released from the paramagnetic beads by MluI digestion. In practice, this method was highly successful in reducing the proportion of nonspecific amplification products, as was shown by both sequencing of random PCR products and restriction digests of PCR products that had been subjected to a second round of amplification
Microarray analysis generally requires labeling of microgram quantities of DNA; however, the DNA yield from a linker-mediated PCR performed on only 1–10 ng of starting material is generally no greater than approximately 200 ng. To address the need for more DNA, we performed a second round of amplification with isothermal MSD, a procedure that provides an enormous degree of amplification while introducing little bias(7); however, MSD amplification has a strong preference for high molecular weight templates(8). To address this issue, we exploited the fact that after the process described above was completed, all PCR fragments had MluI sites at both ends. Self-ligation with T4 DNA ligase produced efficient polymerization of the low molecular weight fragments into higher molecular weight fragments that could be efficiently amplified via MSD
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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.