In this proposal, we will describe a new method and instrument for real-time, target-specific isothermal amplification and DNA quantitation that does not rely on the polymerase chain reaction and does not require expensive fluorescent labeled oligo primers. This new method amplifies the target DNA sequence using the """"""""hairpin loop concatamer DNA amplification"""""""" (HLCA) process, which incorporates self-priming hairpin oligo DNA primers into linear DNA during the initial rounds of replication. Subsequent rounds of replication are primed from these hairpin structures to give isothermal, primer-independent amplification of concatameric DNA structures. The method, if fully realized, has tremendous cost saving and generalized applicability for rapid pathogen and gene quantitation. Data obtained prior to Phase I showed that the HLCA method could amplify trace amounts of target DNA using Bst polymerase at 60 degrees C in one hour. In Phase I, we will optimize this technology for use in our planned instrument and more thoroughly test the use SYBR green (or similar) double-strand DNA fluorescent label dyes for quantitative detection of the newly synthesized amplified DNA. Since the process will produce double-strand DNA molecules, SYBR green dye can be added directly to the amplification reaction and adhere to the newly formed chains. The method will allow us to use a simple, inexpensive visible light detection in a microtiter plate reader or other single beam spectrophotometer for real-time quantitation of the amplified DNA. The instrument and method will significantly reduce the cost and complexity of real-time amplification/detection of pathogens and genes. Based upon our extensive experience in the development of commercially successful, inexpensive and robust instruments for purification of DNA, we believe that the cost per assay will be less than $0.50 with an instrument costing less than $5500. This compares favorably to current technology costing $5-15 per sample and $30,000 to $150,000 per instrument. We will utilize an existing microtiter plate reader, modified for our detection method, for processing up to 96 samples. We will also construct a simplified, rugged field unit for real-time detection of 16 or less samples. An abbreviated version of our automated DNA purification method will be incorporated into the real-time amplification instrument, allowing it to begin with crude samples as one would encounter in pathogen detection work. The product has a $750 million market potential addressing clinical, research and defense applications.
