Wednesday, February 29, 2012

STR Amplification and Detection

STR amplification systems can accommodate a range of template DNA concentrations. Most of the Promega PowerPlex® STR systems provide optimal sister allele balance and locus-to-locus balance with 0.5–1.0ng of DNA template, and studies performed at Promega show that full profiles can be observed with less than 100pg (Ensenberger and Fulmer, 2009; McLaren, 2007; Krenke et al. 2005; Krenke et al. 2002). However, amplification and detection instrumentation can vary. You may need to optimize protocols, including cycle number and detection conditions (e.g., injection time or loading volume), for each laboratory instrument. Most of the PowerPlex® systems use a thermal cycling program with 30 or 32 cycles for 0.5–1ng of purified DNA template. For larger amounts of input DNA (i.e., FTA® paper) or to decrease sensitivity, fewer cycles should be evaluated. In-house validation should be performed to determine the optimal amplification and detection conditions.

The sensitive nature of PCR works in a lab's favor, but it can cause problems if great care is not taken to avoid contaminating the reaction with exogenous DNA. Three main categories of exogenous DNA have the biggest impact on DNA-typing laboratories: 1) DNA from the analyst, 2) DNA from other samples in the lab and 3) allelic ladder fragments. DNA from nonhuman sources, such as bacteria and fungi, will not be amplified and detected because STR systems are species-specific. Extreme care must be taken to avoid cross-contamination when preparing sample DNA, handling primer pairs, assembling amplification reactions and analyzing amplification products. Reagents and materials used prior to amplification should be stored separately from those used following amplification. Amplification reactions should be assembled in a room dedicated for reaction setup, using equipment and supplies reserved for amplification setup. We highly recommend the use of gloves and aerosol-resistant pipette tips. To detect reagent contamination, assemble a negative control reaction (i.e., no template) for every set of reactions, and scrutinize the reactions for the presence of unexpected peaks. After setting up reactions, wash all surfaces with a dilute bleach solution.

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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.