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ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 375 (2008) 373–375
www.elsevier.com/locate/yabio
Notes & Tips
PCR-mediated deletion of plasmid DNA
Mattias D. Hansson *, Kamila Rzeznicka, Matilda Rosenbäck,
Mats Hansson, Nick Sirijovski
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, S-22100 Lund, Sweden
Received 30 October 2007
Available online 8 December 2007
Abstract
The PCR-mediated plasmid DNA deletion method is a simple approach to delete DNA sequences from plasmids using only one
round of PCR, with two primers, and without ligation or purification prior to in vivo recombination. By using only PCR, the method
is sequence independent and, as shown in this study, is applicable to various sizes of plasmids and deletions using minimal primer design.
2007 Elsevier Inc. All rights reserved.
Plasmids often contain DNA that is not needed and,
in some cases, even unwanted. These sequences could
be introns or DNA that has been inserted during the
cloning process. Removal of these inserts previously has
required several time-consuming and costly procedures
such as the introduction of new restriction sites by
PCR and PCR followed by in vitro ligation [1]. Recently,
the SLIM (site-directed, ligase-independent mutagenesis)1
approach presented a viable technique [2]. However, bacteria such as Escherichia coli have the ability to perform
in vivo recombination of DNA with homologous ends
that previously has been used for site-specific mutagenesis
and recombination of DNA constructs [3]. We use this
ability further to remove unwanted DNA from plasmids
using only one round of PCR, with two primers, and
without ligation or purification prior to the in vivo
recombination.
The method was developed in connection with our research on the tetrapyrrole biosynthetic enzymes magnesium chelatase and ferrochelatase [4,5]. Three different
DNA deletions were sought. In one case, the method was
applied to the magnesium chelatase bchH gene of Rhodobacter capsulatus in an attempt to create two specific trunca*
Corresponding author. Fax: +46 46 2224116.
E-mail address: mattias.hansson@biochemistry.lu.se (M.D. Hansson).
1
Abbreviations used: SLIM, site-directed, ligase-independent mutagenesis; SOE, gene splicing by overlap extension; LB, Luria–Bertani.
0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2007.12.005
tions, resulting in one N-terminal domain and one Cterminal domain of the protein for independent functional
characterization. The 9290-bp expression plasmid
pET15bBchH [6] was used as a template to generate the
truncated versions of the magnesium chelatase BchH protein (Table 1). Fragments of 1383 and 2202 bp were deleted
from pET15bBchH to generate the N- and C-terminal domain constructs, respectively. The resulting plasmids were
named pET15bBchHNdom and pET15bBchHCdom. In
the third construct, related to our work on ferrochelatase,
115 bp were deleted from the 3040-bp plasmid pCRNA
to remove a ribosome binding site. The resulting plasmid
was named pCRNA–RBS.
Primer A was designed as the reverse complement of a
sequence corresponding to 16 to 20 bases upstream of
the plasmid DNA to be deleted followed by 16 to 20
bases equal to the downstream sequence (Table 1 and
Fig. 1). Primer B was designed in the same way but corresponded to the complementary strand; that is, the sequence of primer B was identical to the plasmid
primary sequence lacking the sought deletion. A similar
design of primers was employed previously in, for example, the SOE reaction (i.e., gene splicing by overlap
extension) [7]. Primers were added to a 50-ll PCR mixture to a final concentration of 0.2 lM each. Template
of 50 ng and dNTP mix to a final concentration of
0.2 mM of each base was added. Pfu DNA polymerase
was used according to the manufacturer’s instructions
374
Notes & Tips / Anal. Biochem. 375 (2008) 373–375
Table 1
Bacterial strains, plasmids, and primers
Bacterial strain,
plasmid, or
primer name
Strain
E. coli XL1–Blue
Plasmids
pET15bBchH
pCRNA
Primers
fwd-N-domain
rv-N-domain
fwd-C-domain
rv-C-domain
SSremmpfor
SSremmprev
Genotype or description
Reference
recA1 endA1 gyrA96 thi-1
hsdR17supE44 relA1 lac
[F 0 proAB lacIqZDM15
Tn10 (Tetr)]
Stratagene
pET-15b derivative, bchH gene
cloned, 9290 bp in total
pUC19 derivative, 360 bp insert using
the cloning sites KpnI and SacI,
3040 bp in total
[6]
5 0 -AGGGCGCGCTGATCACCG
AATGAGATCTGGCTGCTA-3 0 b
5 0 -GTTAGCAGCCAGATCTC
ATTCGGTGATCAGCGCGC-3 0 b
5 0 -TGGTGCCGCGCGGCAGC
CATGGCCTGCATGTCGTCG-3 0 b
5 0 -CGACGACATGCAGGCCAT
GGCTGCCGCGCGGCACCA-3 0 b
5 0 -CAACCAACCGTGCGCC
CAATTTCATTTTACTCCTCC-3 0 b
5 0 -GAGTAAAATGAAATTG
GGCGCACGGTTGGTTGGGG-3 0 b
a
to recover for 1 h in 1 ml of LB (Luria-Bertani) medium
[8] at 37 C on a rotary shaker (200 rpm). The transformation mixture was plated onto Difco Tryptose Blood
Agar Base (BD Diagnostics, Sparks, MD, USA) plates
(200 ll transformation mixture/plate) containing 100 lg/
ml ampicillin.
The three different PCR-mediated plasmid DNA deletion experiments all resulted in transformants at the first attempt: 4 transformants of E. coli/pET15bBchHNdom, 6
transformants of E. coli/pET15bBchHCdom, and 34 trans-
a
a
a
a
a
a
a
Current work.
Nonunderlined and underlined parts of primer correspond to
sequences upstream and downstream of the plasmid DNA to be deleted.
b
with the 10· Pfu buffer containing 20 mM MgSO4 (Fermentas, St. Leon-Rot, Germany). The PCR was carried
out in a PTC-200 DNA engine (MJ Research, Watertown, MA, USA). The respective annealing and elongation temperatures for the three different reactions were
as follows: pET15bBchHNdom, 43 and 68 C;
pET15bBchHCdom, 55 and 68 C; pCRNA–RBS, 43
and 68 C. Elongation time was adjusted to the length
of the plasmid minus the unwanted sequence, that is,
16, 18, and 7 min (extension rate: 0.5 kb/min) for the
constructs pET15bBchHNdom, pET15bBchHCdom, and
pCRNA–RBS, respectively. PCR was performed for 16
to 18 cycles throughout.
The PCR was followed by DpnI digestion to degrade
unwanted template plasmid prior to transformation.
MgSO4 was added to a final Mg2+ concentration of
10 mM in the 50-ll PCR mixture before adding 10 units
of DpnI (Fermentas). The digestion was performed at
37 C for 2 h. After digestion, 0.5 to 1.5 ll of the mixture
was used to transform 50 ll of electrocompetent E. coli
XL1–Blue cells (Stratagene, La Jolla, CA, USA). The
preparation of the E. coli cells and the electroporation
were performed in accordance with the instructions for
the Bio-Rad E. coli Pulser (Bio-Rad Laboratories, Hercules, CA, USA). The transformed cells were allowed
Fig. 1. Schematic description of the PCR-mediated plasmid DNA
deletion method. The template plasmid shows the DNA to be deleted in
black. The complementary features of the A and B primers and the
plasmid are indicated by gray and striped segments. The steps resulting in
the final construct are indicated by arrows.
Notes & Tips / Anal. Biochem. 375 (2008) 373–375
formants of E. coli/pCRNA–RBS. No efforts were made
to optimize the PCR reactions to increase the number
of transformants. All of the pET15bBchHNdom and
pET15bBchHCdom plasmids and 16 of the pCRNA–
RBS plasmids were isolated and analyzed by restriction enzyme digestion. Of the screened pET15bBchHNdom,
pET15bBchHCdom, and pCRNA–RBS transformants, a
correct cleavage pattern was displayed by 25, 17, and
38%, respectively. All of the plasmids displaying an incorrect cleavage pattern were identified as template plasmids.
All of the sequenced clones displayed the desired sequence
where 1383, 2202, and 115 bp, respectively, had been deleted from the plasmids. No additional unwanted modifications were identified.
We have shown that our method is applicable for plasmids of 3040 bp up to 9290 bp, with deletions ranging from
115 to 2202 bp. Design of the primers was straightforward
and corresponded simply to 16 to 20 bases upstream and
downstream of the plasmid DNA to be deleted. The template plasmid was degraded using DpnI digestion, and the
mixture was then used to transform electrocompetent cells
without further purification.
In summary, we have designed a straightforward and
robust method that is intended to simplify routine molecular biology work carried out on a regular basis in most
laboratories. By using PCR and the ability of bacteria to
perform in vivo recombination of DNA with homologous
ends to delete plasmid DNA, our method is sequence
independent and, as we showed, is applicable to various
sizes of plasmids and deletions using only minimal primer
design.
375
Acknowledgments
K.R. and N.S. are grateful for fellowships from the
Wenner–Grenska Samfundet and the Sven and Lilly Lawski Foundation, respectively. M.H. acknowledges financial
support from the Swedish Research Council.
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