Removal of contaminating TEM-la beta-lactamase gene from commercial Taq DNA polymerase.

Abstract

This study confirms that Taq DNA polymerase could be contaminated with the blaTEM-1(a) gene. It also proposes two different methods that could be used to overcome DNA contamination: (i) DNase I treatment prior to PCR amplification; and (ii) the use of a highly purified Taq DNA polymerase which was devoid of detectable contamination.

Full text

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The Journal of Microbiology, February 2006, p.126-128
Copyright ⓒ 2006, The Microbiological Society of Korea
Vol. 44, No. 1
NOTE
Removal of Contaminating TEM-la β-Lactamase Gene from
Commercial Taq DNA Polymerase
Jae Seok Song1, Jung Hun Lee1,2, Jung-Hyun Lee2, Byeong Chul Jeong1, Won-Keun Lee1 and Sang Hee Lee1,*
1Department of Biological Sciences, Myongji University, San 38-2 Namdong, Yongin, Gyeonggido, 449-728, Republic of Korea
2Marine Biotechnology Center, Korea Ocean Research & Development Institute, Ansan P.O. Box 29, Seoul 425-600,
Republic of Korea
(Received October 21, 2005 / Accepted November 30, 2005)
This study confirms that Taq DNA polymerase could be contaminated with the blaTEM-1a gene.
It also proposes two different methods that could be used to overcome DNA contamination: (i)
DNase I treatment prior to PCR amplification; and (ii) the use of a highly purified Taq DNA pol-
ymerase which was devoid of detectable contamination.
Keywords: Decontamination, DNase I, Taq DNA polymerase, TEM-type β-lactamase
✽ To whom correspondence should be addressed.
(Tel) 82-31-330-6195; (Fax) 82-31-335-8249
(E-mail) sangheelee@mju.ac.kr
β-Lactamases produced by bacterium are known to
protect against the lethal effect of β-lactam anti-
biotics (penicillins, cephalosporins, carbapenems or
monobactams) on cell-wall synthesis. The production
of β-lactamase is the single most prevalent mecha-
nism responsible for the resistance to β-lactams
among clinical isolates of Pseudomonas aeruginosa
and the family Enterobacteriaceae (Sanders and
Sanders, 1992). Extended-spectrum
(ESBLs) are clavulanate-susceptible enzymes confer-
ring broad resistance to penicillins, aztreonam, and
cephalosporins (with the exception of cephamycins)
(Livermore, 1995). ESBLs are often plasmid-medi-
ated, and most of them are mutants of the classic
TEM- and SHV-type enzymes such as TEM-1,
TEM-2, and SHV-1, with one or more amino-acid
substitutions around the active site (Paterson et al.,
2001). These changes allow the hydrolysis of ex-
tended-spectrum cephalosporins (e.g., ceftazidime and
cefotaxime) and monobactams (e.g., aztreonam),
which are stable to the classic TEM- and SHV-type
enzymes (Bradford, 2001). The standard method for
determining the specific gene for more than 130
TEM-type and more than 50 SHV-type ESBLs
(http://www.lahey.org/studies/webt.asp) is the PCR
(polymerase chain reaction) technique followed by
β-lactamases
nucleotide sequencing (Bradford, 2001).
The standard method was performed to detect ESBL
genes from clinical isolates of Enterobacteriaceae in our
nationwide survey (Jeong et al., 2004) and our environ-
mental metagenomic libraries (Song et al., 2005). On
the basis of DNA sequencing of the PCR products for
TEM-type β-lactamase genes, the blaTEM-1a gene was
frequently detected together with other TEM-type
β-lactamase genes such as blaTEM-1b in our survey.
However, this did not occur in our survey when the
other β-lactamase genes such as blaSHV, blaCTX-M,
blaOXA, and blaPER were targeted. The blaTEM-1a gene
was presented in pBR322 (Sutcliffe, 1978) and has
been the most commonly used selective maker for ex-
pression vectors that are generally presented in multi-
ple copies. Thus, it is likely that during Taq DNA
polymerase purification, the DNA harboring blaTEM-1a
gene was not completely removed. To verify this pos-
sibility and avoid cross-contamination, separated rooms
for sample preparation, PCR analysis, and agarose gel
electrophoresis were used. The PCR condition (30 cy-
cles) and DNA sequencing methods were as pre-
viously described (Jeong et al., 2004). Results of PCR
and DNA sequencing indicated that pipette tips (Sarstedt,
Germany), microcentrifuge tubes (Sarstedt, Germany),
and PCR reagents were not the source of blaTEM-1a gene
contamination. When TEM-type β-lactamase genes
were targeted with a TEM primer pair (expected PCR
product size: 839 bp), the contamination in the PCR
reaction was strongly related to the Taq DNA poly-

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Vol. 44, No. 1TEM-1a β-lactamase gene decontamination127
(A)
(B)
Fig. 1. PCR amplification (30 cycles) using the primer pair derived
from TEM-1 β-lactamase gene and commercial Taq DNA polymer-
ase from company A (A) and company B (B). PCR products were
run on a 1% agarose gel with 0.5x Tris-acetate-EDTA buffer. Lane
M, 100-bp plus DNA size marker (sizes in base pairs are indicated
on the left edge of the gel); lane 1, Taq DNA polymerase with ge-
nomic (template) DNA from a clinical isolate producing TEM-1b
and without DNase I treatment; lanes 2-8, Taq DNA polymerase
with the template DNA after treatment of DNase I the amount of
which is 0.05, 0.075, 0.1, 0.25, 0.5, 0.75 or 1.0 unit, respectively;
lane 9, Taq DNA polymerase with double-distilled water instead of
the template DNA and without DNase I treatment; lanes 10-16,
Taq DNA polymerase with double-distilled water instead of the
template DNA after treatment of DNase I the amount of which is
0.05, 0.075, 0.1, 0.25, 0.5, 0.75 or 1.0 unit, respectively. The ar-
row on the right edge of the gel indicates the amplified blaTEM-1b
(lanes 1-8) and blaTEM-1a (lanes 9-12) fragments (837 bp).
merase itself (Fig. 1A). These results were confirmed
from a previous report (Chiang et al., 2005). Contrary
to our study, this report did not show the information
about the subgroup (e.g., blaTEM-1a, blaTEM-1b, and so
on) of contaminated blaTEM-1 gene and about any
method necessary for overcoming the blaTEM-1a gene
contamination of the Taq DNA polymerase. Using the
negative control (double-distilled water), the Taq
DNA polymerase from company A produced a
false-positive signal, but the Taq DNA polymerase
highly purified by company B did not (Fig. 1B). The
Taq DNA polymerase has a high affinity for DNA,
and thus a certain amount of contaminating DNA
such as the blaTEM-1a gene may always remain pro-
tected from physical or chemical treatment (Corless et
al., 2000). PCR is a highly sensitive technique widely
used for the rapid detection of specific DNA se-
quences, with numerous applications in genotyping of
β-lactamase genes, clinical diagnosis, and microbial
identification (Lee et al., 2005). These PCR techni-
ques can amplify a single copy of template DNA 106-
to 107-fold, and thus small amounts of exogenous
DNA is a limitation to PCR approaches (Corless et
al., 2000; Newsome et al., 2004). The identification
of exogenous blaTEM-1a gene contamination in Taq
DNA polymerase must be important to the inves-
tigators, especially those who work on TEM-type
ESBLs. The tainted Taq DNA polymerase can pro-
duce false-positive results that can be extremely con-
fusing and misleading. It can also be very time-con-
suming for investigators to follow up on the
false-positive products. Thus, some strategy is neces-
sary for overcoming the blaTEM-1a gene contamination
of Taq DNA polymerase.
Since the most effective method for the elimination
of exogenous DNA was a DNase I treatment (Corless
et al., 2000; Tondeur et al., 2004), RQ1 RNase-Free
DNase (Promega, USA) was added to the PCR re-
action mixture without the primer set. For each 13 μl
of PCR mixture, 0.05, 0.075, 0.1, 0.25, 0.5, 0.75, or
1.0 unit of the enzyme was added together with 2 μl
of its specific buffer. The mixture solution was in-
cubated for 30 min at 37°C, then for 10 min at 65°C
after the addition of 2 μl of the RQ1 DNase Stop
Solution (Promega, USA) to inactivate the DNase I.
After the addition of 2 μl primer set and 1 μl tem-
plate DNA (or double-distilled water) into the mix-
ture, a PCR was performed. The best results for over-
coming the blaTEM-1a gene contamination were ob-
tained with 0.25 unit of the DNase I for 13 μl of
PCR mixture (Fig. 1A). This amount of DNase I was
sufficient to eliminate any trace of contaminated
blaTEM-1a genes from the PCR mixture without altering
the efficiency of the PCR reaction (Fig. 1A). These re-
sults were highly reproducible in several experiments.
The presence of DNA contamination surrounding
Taq DNA polymerase was previously reported in sev-
eral PCR studies (Böttger, 1990; Rand and Houck,
1990; Schmidt et al., 1991; Corless et al., 2000;
Hughes et al., 2000; Newsome et al., 2004). Most of
the contamination reported was exogenous bacterial
DNA. In all of these previous reports, PCR amplifica-
tion was performed with universal primers for the
highly-conserved 16S rDNA gene, whereas in the
present study, amplification was done with primers
targeting the TEM-type β-lactamase gene. Here we
have revealed that standard preparations of Taq DNA
polymerase are contaminated with blaTEM-1a gene and
proposed two methods to efficiently detect ESBL
genes from clinical isolates by a standard PCR, using
either decontamination with low concentrations of
DNase I prior to PCR amplification or a highly puri-
fied Taq DNA polymerase without exogenous DNA.
These methods that overcome the blaTEM-1a gene con-
tamination of Taq DNA polymerase were highly
reproducible. They could be useful in microbial stud-
ies where the presence of contaminating exogenous

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128 Song et al.J. Microbiol.
DNA is a limitation to PCR approaches.
Acknowledgement
This work has been supported by a research grant
from BioGreen 21 Program (20050301034479), Rural
Development Administration, Republic of Korea, by
the Driving Force Project for the Next Generation of
Gyeonggi Provincial Government, and by in-house
program (PE87200) of KORDI.
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