Biomedical Engineering Reference
In-Depth Information
Chapter 1
P
RODUCTION OF
T
HERMOSTABLE
DNA
P
OLYMERASE
S
UITABLE FOR
W
HOLE
-B
LOOD
P
OLYMERASE
C
HAIN
R
EACTION
A. S. Korovashkina, S. V. Kvach, L. A. Eroshevskaya
and A. I. Zinchenko
*
Institute of Microbiology, National Academy of Sciences, Minsk, Belarus
A
BSTRACT
Using methods of genetic engineering the chimeric protein with additional N-
terminal sequence-non-specific DNA binding domain of
Sulfolobus solfataricus
(SSO7D)
was obtained on the basis of Klen
Taq
DNA polymerase (N-terminal 279 amino acids
deletion variant of full length
Taq
DNA polymerase). Fermentation, isolation and
purification of the target enzyme was performed in selected conditions. It was found that
SSO7D-Klen
Taq
DNA polymerase retained stability at elevated blood concentrations (up
to 20%, v/v) and it can be used as a component of analytical kits in clinical
investigations.
Keywords:
Polymerase chain reaction, plasmid, induction of protein expression, pET system,
SSO7D-Klen
Taq
DNA polymerase, sequence-non-specific DNA binding domain,
Escherichia coli
1.
I
NTRODUCTION
Polymerase chain reaction (PCR) is a simple, efficient, and economical method for
amplifying a small amount of DNA template but purified DNA is required [1, 2]. Blood as a
typical biological sample is widely used in PCR-based studies, in diagnosis of microbial
infections and genetic diseases, as well as in forensic analyses and blood banking [3, 4].
