ALLELE-SPECIFIC MULTIPLEX DNA DIAGNOSTIC TEST

THE AIM OF THIS PROJECT IS TO DEMONSTRATE A NEW MULTIPLEX DNA DIAGNOSTIC TEST WHICH CAN SIMULTANEOUSLY DETECT NUCLEOTIDE DIFFERENCES BETWEEN SEVERAL DIFFERENT ALLELES AT SEVERAL DIFFERENT GENETIC LOCI IN A SINGLE TEST SAMPLE OF HUMAN DNA. THE TEST PORTION OF THE PROTOCOL RELIES ON ALLELE-SPECIFIC POLYMERASE CHAIN REACTIONS (PCR) TO PRODUCE AMPLIFIED PRODUCTS ONLY WHEN THE PRIMER SEQUENCES ARE COMPLEMENTARY TO THE LOCUS BEING TESTED. MANY OF THESE ALLELE-SPECIFIC PCRS WILL BE CARRIED OUT SIMULTANEOUSLY ON A SINGLE TEST SAMPLE OF DNA. THE EXISTENCE OF AMPLIFICATION PRODUCTS FROM THE VARIOUS PRIMERS WILL THEN BE DETERMINED BY MULTIPLEX CAPTURE OF THE PRODUCTS ON A SOLID SUPPORT BY HYBRIDIZATION BETWEEN SPECIFIC OLIGONUCLEOTIDES WHICH WERE PREVIOUSLY ATTACHED TO THE SUPPORT. RESULTS WILL BE READ BY THE PRESENCE OR ABSENCE OF LABELED AMPLIFICATION PRODUCTS AT EACH SPOT ON THE SUPPORT. LABELS WILL BE NONRADIOACTIVE SUCH AS BIOTIN-STREPTAVIDIN-HORSERADISH PEROXIDASE. HYBRIDIZATION TO THE TAIL COMPLEMENTS ON THE SOLID SUPPORT WILL BE BY CONVENTIONAL WATSON-CRICK HYDROGEN BONDING OR BY HOOGSTEEN BONDING IN A TRIPLE HELIX CONFIGURATION. IN THE LATTER CASE, NO DENATURATION OF THE PCR PRODUCTS WILL BE REQUIRED PRIOR TO HYBRIDIZATION. IN THIS NEW TEST, ALLELE-SPECIFIC PCR AMPLIFICATIONS WILL BE PERFORMED IN BATCHES OF ABOUT TEN PER DNA SAMPLE, BUT THE TEST IS EXPECTED TO PERMIT SIMULTANEOUS CAPTURE AND SELECTION OF PRODUCTS FROM HUNDREDS OF ALLELES ON A SINGLE SOLID SUPPORT.