THERMOSTABLE DNA-POLYMERASE, METHOD OF ITS PREPARING, FRAGMENT OF ISOLATED DNA, METHOD OF DNA AMPLIFICATION, METHOD OF LABEL INCORPORATION IN DNA AND METHOD OF REVERSE TRANSCRIPTION

摘要

FIELD: biotechnology, biochemistry, enzymology. SUBSTANCE: DNA-polymerase is prepared from thermophilic eubacterium Carboxydothermus hydrogenoformans or E. coli BL21 (DE3) pUBS520pAR4 DSM No 11179. DNA- polymerase elicits magnesium ions-dependent activity of reverse transcriptase and activity of 3'-5'- exonuclease, its apparent molecular mass is about 100-105 kDa. Invention involves also recombinant plasmids and transformed host-cells that are able to produce enzyme. Fragment of isolated DNA with nucleotide sequence given in description encodes thermostable DNA-polymerase. DNA fragment is prepared from Carboxydothermus hydrogenoformans. Natural strain of Carboxydothermus hydrogenoformans or E. coli BL21 (DE3) pUBS520pAR4 No 11179 is cultured, cells of strain are suspended in buffer and disrupted. DNA- polymerase is purified by chromatography method. In amplification, label incorporation and reverse transcription carrying out thermostable DNA-polymerase is used. Invention provides preparing DNA-polymerase exhibiting activity at higher temperatures and eliciting 3'-5'-polymerase activity and reverse transcriptase activity in the presence of magnesium ions and absence of manganese ions. EFFECT: improved method of preparing, amplification and reverse transcription. 8 cl, 12 dwg