Thermostable DNA polymerase from Thermus thermophilus B35: preparation and study of a modified form of the enzyme with high affinity to ddNTP.
The hybrid protein consisting of Tte DNA polymerase fragment and mutant Taq DNA polymerase (F667Y) fragment in the ratio 20 : 1 was constructed. Affinity of the modified enzyme (substitutions F669Y, V667I, and S692Q) to ddNTP was two orders higher than that of the wild type enzyme. The modified enzyme was used for sequencing DNA fragment with total deoxyguanosine and deoxycytidine content of 68%. In the polymerase chain reaction, the modified enzyme exhibits properties typical of the wild type Tte DNA polymerase.
Akishev
A G
AG
Institute of Molecular Biology and Biophysics, Siberian Branch of the Russian Academy of Medical Sciences, Novosibirsk 630117, Russia.
Rechkunova
N I
NI
Tomilova
J E
JE
Lebedeva
N A
NA
Lavrik
O I
OI
Degtyarev
S Kh
SKh
D001345:Autoradiography; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009705:Nucleosides / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D017931:DNA Primers; D009705:Nucleosides; C093010:DNA polymerase, Thermus thermophilus; D004259:DNA-Directed DNA Polymerase