Biomedical Engineering Reference
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medium: 1% (w/v) tryptone, 0.5% (w/v) yeast extract and 0.5% (w/v) NaCl in deionized
water adjusted to pH 7 with KOH. Cultivation was continued until the optical density at 600
nm reached 0.6. Then isopropyl-β-thiogalactopyranoside (IPTG) was added up to
concentration of 1 mM and fermentation was carried out for another 5 h. Cells were harvested
by centrifugation for 10 min at 12,000 × g, washed once with 30 mM potassium phosphate
buffer (pH 7.0) and slurried in the buffer, consisting of 50 mM NaH
2
PO
4
, 300 mM NaCl and
20 mM imidazole (рН 8.0).
The cells were sonicated twice at 4°C for 2.5 min and heated at 70
о
С for 30 min. After
centrifugation of the sample for 30 min at 20,000 × g, the SSO7D -Klen
Taq
DNA polymerase
was isolated from supernatant, using metal affinity chromatography with Ni-NTA agarose
(Qiagen, USA). The resulting enzyme solution was dialyzed overnight against several
changes of 10 mM Tris-HCl buffer (pH 8.0) containing 100 mM KCl, 1 mM EDTA, 2 mM β-
mercaptoethanol and 0.5% Tween 20.
The dialyzed protein sample was dissolved three-fold in 10 mM Tris-HCl buffer (pH 8.0)
containing 100 mM KCl, 1 mM EDTA, 2 mM β-mercaptoethanol, 0.5% Tween 20 and 50%
glycerol.
The activity of SSO7D-Klen
Taq
DNA polymerase was determined using PCR.
Amplification by PCR was performed in a reaction mixture (30 μl) containing 5 ng of
plasmid DNA as a template, synthetic primers (5 pmol each), F 5'-
GTCTACCAGGCATTCGCTTCAT-3' and R 5'-CTGTGA ATGCTGCGACTACGAT-3'),
four deoxyribonucleoside triphosphates (0.2 mM each), MgCl
2
(3 мМ), KCl (90 mM), 50 mM
Tris-HCl buffer (pH 8.8), 0.05% Tween 20 and 1 μl of SSO7D-Klen
Taq
DNA polymerase
solution. The reaction was conducted for 30 cycles (15 s at 95
о
С, 15 s at 65
о
С and 30 s at
72
о
С). The 3 μl sample of the PCR mixture was then added to 200 μl of DNA-intercalating
dye SYBR Green I (10,0000X stock solution, Sigma, USA) diluted 1:1600 in TE buffer, and
fluorescence was measured using Qubit fluorometer (Invitrogen, USA). The amount of
SSO7D-Klen
Taq
DNA polymerase corresponding to 1 unit of
Taq
DNA polymerase (Sileks,
Russia) was assumed to be equivalent to one unit.
3.
R
ESULTS AND
D
ISCUSSION
It is known from literature reports that SSО7D domain allows to raise considerably
affinity of DNA polymerase to the matrix and, as consequence, to increase processivity,
synthesis rate, resistance to high ionic strength and various inhibitors [8]. Based on these data
we presumed that chimeric DNA polymerase derived in the course of this research will be
also more resistant to whole-blood inhibition. To assess resistance of SSО7D-Klen
Taq
DNA
polymerase to whole blood we performed analysis of single point mutation in gene encoding
blood coagulation factor V (FV, FV Q506, or FV Leiden). Presence in this gene of G→A
substitution at nucleotide 1691 position is reported to bring up risk of venous
thromboembolism in heterozygotes 8-fold, in homozygotes - 80 or even 100 fold [11].
Although only 5% of adult population are carriers of mutant allele, this mutation is detected
in 40% of patients suffering from venous thrombosis. It follows that early screening for this
mutation is essential for accurate diagnosing and formulation of proper treatment strategy. To
evaluate effect of EDTA-stabilized blood on PCR engaging our original SSO7D-Klen
Taq
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