TECHNOLOGY FOR DETECTION OF SINGLE BASE MUTATIONS

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 SUPRESSOR 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 OVEREXPRESSED, INDICATE THAT IT IS CAPABLE OF DETECTING SIGNIFICANTLY MORE SINGLE-BASE MUTATIONS THAN 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 IS ONLY ABLE TO CLEAVE AFTER C, G AND U RESIDUES. IT WILL BE DETERMINED HOW EFFECTIVE RNASE I IS FOR DETECTING ALL OF THE 12 POSSIBLE SINGLE-BASE MISMATCHES. IN ADDITION, REACTION CONDITIONS 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 CLEVAGE WITH CURRENTLY KNOWN RIBONUCLEASES WILL BE DEVELOPED. THIS NEW TECHNOLOGY WILL BE TESTED USING A SERIES OF P53 GENES CONTAINING SINGLE-BASE MUTATIONS.