Research Project
3497795
Non-radioactive DNA based detection systems offer an attractive new approach to the clinical diagnosis of a variety of viral and bacterial pathogens. Current methods for labeling DNA by enzymatic procedures are time consuming, labor intensive, and not well suited for large scale production. This proposal describes a novel method for the chemical non-radioactive labeling of DNA probes which overcomes the problems inherent in enzymatic procedures. This simple, two step procedure involves the attachment of a hydrazide linker arm to cytosine residues, followed by coupling of a reporter molecule to the linker arm. In Phase I we will be determining optimal conditions for (1) linker arm coupling and (2) attachment of several reporter molecules (biotin, Texas red, rhodamine B) to the mononucleotide 2'-deoxycytidylic acid. The most suitable procedures will be employed in the labeling of poly dC and M13 single stranded DNA. The hybridizability and detection sensitivity of these newly constructed probes will be briefly examined. The labeling of other DNA's and more detailed studies on hybridization and sensitivity will be carried out in Phase II. The chemical labeling procedure outlined in this proposal should represent a major advance in the commercialization of DNA based technology.
