NEW METHOD OF NONRADIOACTIVE LABELING OF OLIGONUCLEOTIDES AND THEIR USE AS ALLELE-SPECIFIC PROBES FOR MUTATIONS CAUSING BETA-THALASSEMIA

Schemes of hybridization analysis allowing the genotype to be determin ed in 1 ng of amplified DNA were elaborated to reveal IVS 1-110 mutati ons in the beta-globin gene, causing beta-talassemia. The analysis inc ludes amplification of a beta-globin gene fragment by polymerase chain reaction (PCR) and subsequent hybridization of amplified DNA with all ele-specific probes labeled with biotin or horseradish peroxidase. A s imple, rapid, and efficient method is proposed for covalent enzyme bin ding with oligonucleotide, allowing to obtain conjugates with 60-80% y ield, and to use the conjugates in the hybridization without their ext raction from reaction mixture. The approach suggested allows to simpli fy and to reduce the time of the procedure. The method is recommended for simple and rapid preparation of hybridization probes for different purposes.