RAPID, HIGHLY EFFICIENT SUBTRACTION CDNA CLONING METHOD

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DESCRIPTION (Adapted from the Investigator's Abstract): The identification of the difference between two complex gene pools holds tremendous promise for the discovery of infectious agents, genetic mutations, cell function, and for finding probes that are diagnostic of disease state. Phase I work will create a rapid, highly efficient, method and set of reagents for subtraction cDNA cloning. This method will be at least three times faster and several fold more efficient at removing the common clones between two pools than any subtraction cloning method published to date. The proposed method can be performed on virtually any plasmid or lambda tester and driver libraries or mRNA pools. The method will use a novel and powerful new hybridization technology in which nucleic acids are electrophoresed through boundaries of acrylamide which have covalently bound DNA capture DNA probes. This process of "capture probe electrophoresis" has been shown to rapidly and efficiently remove common sequences due to the high concentration of bound probe and the fact that the process highly concentrates the nucleic acid which hybridizes to the bound capture probe during electrophoresis. The bound capture probe will be designed so that is matches common sequences in amplified driver DNA so that the same acrylamide-oligo reagent can be used for subtraction of nearly any plasmid or lambda cloned sequences. Experiments prior to Phase I show that the proposed process is highly efficient at enriching unique sequences in a tester pool. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE