RESUMO
A method of subtractive hybridization using biotinylated DNA and phenol emulsion reassociation technique (PERT) has been proposed. A possibility of combining these techniques has been shown for the first time. The effect of biotinylation degree on the formation of water-insoluble Bio-DNA aggregates was studied. The conditions when Bio-DNA aggregation is actually absent were revealed. A possible use of the above method in hybridization experiments in a wide range of DNA concentrations has been shown. The time of hybridization was 0.5-1 h. The method was approved on a model system, and its possible application for the enrichment of rare mRNA was shown. No less than 300-fold enrichment is achieved for a rare transcript (IL-2) in three cycles of subtractive hybridization.
Assuntos
Biotinilação , DNA/metabolismo , Hibridização de Ácido Nucleico , Fenol/química , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Humanos , Interleucina-2/biossíntese , Células Jurkat , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
A human interleukin-2 (IL-2) gene was isolated from genomic DNA library. The isolated gene with 5'- and 3'-flanking sequences of various lengths was inserted into plasmids derived from the retroviral vector pPSneo. The recombinant plasmids were transfected into myeloma X63Ag8-653 cells. The transfected cells, harbouring the IL-2 gene with the shortened (to position -165) or totally deleted 5'-flanking sequence, constitutively expressed biologically active IL-2. Deletion of 3'-flanking region on did not affect the IL-2 expression.