The investigator asserts that the principal inhibitor of TAQ polymerase in blood samples is heme. The goal of the revised proposal is to develop chemical, photochemical, enzymatic, or affinity binding methods for the inactivation of heme with a long term objective of allowing PCR to become a routine clinical diagnostic tool with minimal handling of the samples to be amplified: For example, whole blood samples without purification of peripheral blood mononuclear cells or dry blood smears which have existed for extended periods of time. The methods to be assayed are the inactivation of heme by direct chemical or photochemical procedures, the inactivation of heme by enzymatic procedures and the removal of heme by affinity binding procedures. Heme is reported to totally inhibit PCR at a concentration of o.8 micromolar. It becomes a major problem in situations with low copy number of pathogens since large volumes of blood are required for sample preparation and hemolysis must be avoided in the procedure. Attempts to overcome this inhibitory effect have been by adding more template or by the addition of globin to bind the heme or in other studies by the additional quantities of TAQ polymerase or primer. The proposal here however, will pursue the inactivation or removal of the heme. In order to minimize the number of procedures to be carried out, the major conversion products where commercially available, will be obtained and assayed for their effect on the model PCR system. For chemical inactivation, reductive and oxidative procedures will be assayed working on the porphyrin ring or alteration of the iron atom through valance change, removal or substitution. Photochemical changes induced by irradiation of whole or partially processed blood are expected to produce oxidation products of heme and it will be determined if these oxidation products of themselves are inhibitory. Since heme is naturally degraded to biliverdin and bilirubin, if these have inhibitory effects exposure to visible light not only alters the binding of bilirubin to albumin but induces photo degradation. Again, these photo products will be examined for inhibitory effects. In addition, the effects of these procedures on the nucleic acids will be examined. The enzymatic inactivation of heme will be followed by employing heme oxygenase and again the reaction products bilirubin and biliverdin, will be assessed in this reaction. Finally, the removal of heme by affinity binding methods will be undertaken. Here apparently the attempt will be made to fill the axial ligands of iron to prevent binding between heme and the TAQ polymerase and also an attempt will be made to bind the iron with transferrin (a reaction known not to occur) globin or myoglobin (really apomyoglobin). Deferoxamine will also be attempted, however, this is another agent known not to bind to heme iron or to remove iron from heme. All these attempts will be assayed in a model system using standard HLA-DQalpha PCR reaction.
