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 determined in 1 ng of amplified DNA were elaborated to reveal IVS 1-110 mutations in the beta-globin gene, causing beta-talassemia. The analysis includes amplification of a beta-globin gene fragment by polymerase chain reaction (PCR) and subsequent hybridization of amplified DNA with allele-specific probes labeled with biotin or horseradish peroxidase. A simple, rapid, and efficient method is proposed for covalent enzyme binding with oligonucleotide, allowing to obtain conjugates with 60-80% yield, and to use the conjugates in the hybridization without their extraction from reaction mixture. The approach suggested allows to simplify and to reduce the time of the procedure. The method is recommended for simple and rapid preparation of hybridization probes for different purposes.