DNA PROBES TO DETECT PREDISPOSITION TO TYPE I DIABETES

INSULIN-DEPENDENT DIABETES MELLITUS (TYPE I) OCCURS IN ABOUT0.5% OF CAUCASIANS. THE INCIDENCE INCREASES OVER 20-FOLD ININDIVIDUALS WITH THE SPECIFIC SUSCEPTIBILITY GENE HLA-DQ3.2. APPROXIMATELY 70% OF INDIVIDUALS WHO BECOME INSULIN-DEPENDENT DIABETICS CARRY THIS GENE, MAKING IT THE MOST SPECIFIC GENETIC MARKER KNOWN FOR IDENTIFYING INDIVIDUALS AT RISK. STANDARD METHODS ARE INADEQUATE FOR IDENTIFICATION OF THE DQ3.2 GENE. HOWEVER, DNA OLIGONUCLEOTIDE PROBES HAVE BEEN USED IN SOUTHERN BLOTS WITHGENOMIC DNA TO IDENTIFY THE DQ3.2 GENE, AND TO DISCRIMINATE IT FROM CLOSELY RELATED, NON-DIABETOGENIC ALLELES. IN THIS WORK, DNA OLIGONUCLEOTIDE PROBES WILL BE USED TO DETECT BY IN SITU HYBRIDIZATION THE DQ3.2 MRNA TRANSCRIBED IN HUMAN LYMPHOCYTES. ENZYME-LABELED DNA OLIGONUCLEOTIDE PROBES WILLBE HYBRIDIZED AGAINST LYMPHOCYTES FIXED ON GLASS SLIDES. HYBRIDIZATION WITH TARGET DQ3.2 MRNA WILL BE DETECTED BY VISUALIZATION OF A COLORED PRECIPITATE. PROCEDURES WILL BE DEVELOPED FOR RAPIDLY FIXING MRNA WITHIN CELLS, AND FOR DETECTING DQ3.2 MRNA WITH HIGH SPECIFICITY AND SENSITIVITY. THE LONG-TERM OBJECTIVES ARE TO DEVELOP A PRODUCT FOR THE DETECTION OF GENETIC PREDISPOSITION TO TYPE I DIABETES, AND A RAPID AND SENSITIVE IN SITU TECHNOLOGY FORTHE DETECTION OF ADDITIONAL GENETIC MARKERS.