The goal of this project is to develop a ribonuclease protection/mismatch assay, which will allow the detection of essentially all single-base mutations. Such an assay would provide an inexpensive and widely applicable method for screening for genetic diseases, detecting mutations in oncogenes and tumor suppressor genes,typing viruses and for other research and diagnostic applications. Current ribonuclease protection assay procedures are able to detect only about two-thirds of single-base mutations. Preliminary results with RNase I from E.coli, which has recently been cloned and over expressed, indicate that it's capable of detecting significantly more single-base mutations than can the conventionally used RNases A and T1. RNase I is able to cleave after all four bases, unlike the combination of RNase A and T1 which are only able to cleave afterC, G and U residues. It will be determined how effective RNase I is at detecting all of the 12 possible single-base mismatches. In addition, reactionconditions for detection of single-base mismatches will be optimized, and, if necessary, an assay which will rapidly identify in crude cell homogenates novel ribonucleases with the ability to cleave at single-base mismatches refractory to cleavage with currently known ribonnucleases will be developed. This new technology will be tested using a series of p53 genes single-base mutations.
