Ionic liquids promote PCR amplification of DNA

Article abstract of DOI:10.1039/c2cc31740k

A bicyclic imidazolium ionic liquid (4d), [b-4C-im][Br], was found to be highly effective not only for promoting PCR of GC-rich DNA by minimizing non-specific amplification, but also for facilitating PCR of normal-GC DNA under mild conditions.

Full text of DOI:10.1039/c2cc31740k

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COMMUNICATION
Ionic liquids promote PCR amplification of DNAw
Yugang Shi,ab Yen-Liang Liu,a Peng-Yeh Lai,c Ming-Chung Tseng,a Min-Jen Tseng,c
Yudong Lib and Yen-Ho Chu*a
Received 8th March 2012, Accepted 5th April 2012
DOI: 10.1039/c2cc31740k
A bicyclic imidazolium ionic liquid (4d), [b-4C-im][Br], was found
to be highly effective not only for promoting PCR of GC-rich
DNA by minimizing non-specific amplification, but also for
facilitating PCR of normal-GC DNA under mild conditions.
the detection and diagnosis of diseases, the forensic identi-
fication of genetic fingerprints, and the creation of transgenic
organisms. One major problem, however, commonly associated
with PCR experiments that limited the output of its routines is
their poor to nil amplification of GC-rich DNA sequences
under standard reaction conditions. This was primarily due to
the spontaneous formation of secondary structures in DNA.3
Many eukaryotic genes such as transcriptional regulatory
elements present in the promoter, enhancer, and locus control
regions that critically regulate gene expression are GC-rich
DNA.5 In the literature, the aforementioned PCR obstacle
could be improved either by using modified reaction protocols
such as ‘‘slowdown PCR’’3 or by adding PCR enhancing reagents6
such as DMSO and betaine. Their capacities to improve
performance in PCR experiments in some cases, however, were
marginal.3,6 We envisioned that the tunable structure and high
polarity of ionic liquids should allow us to discover new ionic
liquid-based enhancers and, therefore, decided to explore PCR
by using ionic liquids to ameliorate the amplification of GC-rich
DNA sequences. In this report, we studied PCR amplification
of two gene fragments, A (266 bp, 80% GC content) and
B (501 bp; 55% GC content), of Streptomyces coelicolor
genomic DNA,7 using protocols that included ionic liquids
1a–f, 2a–f, 3a–f, and 4a–f (Fig. 1). Details of DNA sequences
for primers and templates are summarized in ESI-1.w To our
knowledge, this use of ionic liquids as enhancing reagents for
PCR amplification of DNA templates has not been described.
We totally examined twenty four ionic liquids (for synthesis
and characterization of all ionic liquids studied, see ESI-2w).
Using ‘‘slowdown PCR’’,3 the effectiveness of these ionic
liquids was screened and the result of PCR amplification of
the GC-rich, 266 bp gene fragment A is summarized in Fig. 2
(for gel results, see Fig. S1–S4 in ESI-1w) (for PCR condi-
tions, see Materials and Methods in ESI-1w). We found that
This communication reports a new application of ionic liquids
for use in facilitating the polymerase chain reactions (PCR) of
DNA. Ionic liquids are low-melting molten salts composed entirely
of ions, and many of them are liquid at room temperature.1 Ionic
liquids carry numerous desirable properties such as wide liquid
range, thermal and chemical stability, remarkable solubility
with many small molecules, high polarity and conductivity,
attractive recyclability, and negligible vapor pressure that are
well suited for a myriad of innovative applications, including
reaction media for organic synthesis, chemical sensing and
catalysis, affinity separation, electrolytes for solar and fuel cells,
high temperature lubricants, and re-writable image surfaces.1
In our laboratory, we have been interested in developing
new ionic liquids and have an ongoing program to evaluate
ionic liquids as novel and stable media for chemical and
biochemical applications.1a,1e,2 Moreover, we have recently
employed affinity ionic liquid (AIL)2d and sensing ionic liquid
(SIL)2c to explore functionalized ionic liquids through incor-
poration of functional groups as a part of developing ionic
liquids possessing tailored properties. Herein, we present ionic
liquids as a new class of enhancers that can be used directly to
promote PCR by eliminating non-specific amplification of DNA.3
Since its invention in 1988, PCR has become the most widely
used molecular biology technique that employs thermophilic
polymerase enzyme for exponentially amplifying segments of
DNA.4 Today, PCR is routinely used in medical and biological
applications such as the sequencing and replication of genes,
a Department of Chemistry and Biochemistry,
National Chung Cheng University, Minhsiung, Chiayi 62102,
Taiwan, ROC. E-mail: cheyhc@ccu.edu.tw; Fax: +886 52721040;
Tel: +886 52428148
b College of Food Science and Biotechnology, Zhejiang Provincial Key
Laboratory of Food Safety, Zhejiang Gongshang University,
Hangzhou, Zhejiang 310035, PR China
c Institute of Molecular Biology, National Chung Cheng University,
Minhsiung, Chiayi 62102, Taiwan, ROC
w Electronic supplementary information (ESI) available: Fig. S1–S11
and experimental procedures of PCR with ionic liquids (ESI-1, 22 pages);
synthesis, NMR spectra and spectral data of all ionic liquids (1a–f, 2a–f,
3a–f and 4a–f) studied in this work (ESI-2; 59 pages). See DOI: 10.1039/
c2cc31740k
Fig. 1 Twenty four ionic liquids, [R-mim][X] (1a–f), [R-dmim][X] (2a–f),
[R-3C-im][X] (3a–f), and [R-4C-im][X] (4a–f), used in this work.
c
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able to amplify successfully this 266 bp template A. Nonspecific
gene amplification was evident in the absence of additives
(lane C, Fig. 3). It was reported in the literature that DMSO
and betaine required much higher concentrations to proceed
specific gene amplification.6,8 In our hand, both DMSO and
betaine at 70 mM (for DMSO, 70 mM approximates 0.5%, v/v)
were found totally ineffective (lanes 4 and 5, Fig. 3). Since
ionic liquids are organic salts, it could be argued that they
provide nothing more than the increase of salt concentration
in buffer for PCR. However, no PCR product A was observed
if only KBr was substituted for [b-4C-im][Br] 4d (lane 3,
Fig. 3), suggesting a more direct interaction between the ionic
liquid and the PCR system.
Fig. 2 Screening of ionic liquids (70 mM) used to promote PCR
amplification of a GC-rich (80% GC content), 266 bp gene fragment A.
We also studied the effect of ionic liquids (70 mM each) on
the PCR amplification of DNA with normal GC content, the
template B (55% GC, 501 bp). Here, the PCR protocol used
for amplifying the template DNA B was: 24 cycles of dena-
turation at 95 1C for 20 s, annealing at 57 1C for 30 s, and
extension at 72 1C for 30 s. The result is summarized in Fig. 4.
Under our experimental conditions, an additive was clearly
required for specific amplification of the gene fragment B; that
is, without ionic liquids, no PCR product B was obtained
(controls in Fig. 4) (lanes C in Fig. S6–S9, ESI-1w). To our
delight, a number of ionic liquids were found to promote this
PCR of normal GC, 501 bp DNA B (for gel results, see
Fig. S6–S9, ESI-1w). Among them (1d–e, 2d–e, 3c–e, and 4c–d),
ionic liquids having a butyl group (1d, 2d, 3d, and 4d) all
promoted the PCR of the gene fragment B (R = C4H9, Fig. 4)
(Fig S10, ESI-1w). In our hands, common PCR enhancing
reagents (DMSO and betaine) and the KBr salt, 70 mM each,
were totally unable to direct the amplification of B, and only
ionic liquid (4d) promoted PCR amplification of DNA B,
suggesting that ionic liquid 4d is far more superior in specific
PCR amplification of GC-rich as well as normal GC DNA
templates (Fig. 5).
the amplification of gene fragment A was completely dependent
on the presence of additives; that is, without ionic liquids, no
PCR product A (but only nonspecific DNA amplification) was
resulted (controls in Fig. 2) (lanes C in Fig. S1–S4, ESI-1w).
Smeared nonspecific background bands in gels reflected a high
GC content of DNA (Fig. S1–S4, ESI-1w).3,6 Under our
experimental conditions with ionic liquids at its low concen-
tration (70 mM),8 a bicyclic imidazolium [b-4C-im] cation (4d)
is clearly required for effective amplification of this GC-rich
DNA template A (Fig. 2). Monocyclic imidazolium cations,
[R-mim] (1a–f) and [R-dmim] (2a–f), and five-membered
bicyclic imidazolium [R-3C-im] (3a–f) cations practically gave
no PCR adduct A (Fig. S1–S3, ESI-1w). Moreover, in the case
of six-membered bicyclic imidazolium [R-4C-im] (4a–f)
cations, the length of the alkyl chain appears to be important;
both propyl (4c) and butyl (4d) groups gave specific PCR
product A, but with insignificant amplification using 4c, and
methyl (4a), ethyl (4b), pentyl (4e), and hexyl (4f) produced
no adduct A at all (Fig. S4, ESI-1w). When ionic liquids
with the same butyl chain length were compared (1d, 2d, 3d,
and 4d), only the 6-membered bicyclic ionic liquid 4d effec-
tively promoted the PCR amplification of gene fragment A
(Fig. S5, ESI-1w).
This PCR enhancement by ionic liquids prompted us to
investigate their possible role in lowering melting temperatures
(Tm) of DNA duplexes. First, PCR products A and B were
prepared and used to measure their fluorescence annealing
curves (ESI-1w). The results for GC-rich PCR adduct A shown
in Fig. 6 clearly indicate that the lowest annealing temperature
(Tm = 90.5 1C) was measured for DNA with the ionic liquid
4d and the highest value (Tm = 94.1 1C) was obtained for
DNA with no ionic liquid or DMSO. At 70 mM, DMSO
appeared to be insignificant in lowering the Tm under our
Encouraged as we were by the PCR result, we then turned
our attention to compare this new PCR enhancer 4d with two
commonly employed enhancing reagents DMSO and betaine,6
and investigated their effectiveness on specific PCR amplification
of the template A. The result shown in Fig. 3 clearly demon-
strated that, among all additives tested, only ionic liquid 4d was
Fig. 3 PCR amplification of GC-rich 266 bp DNA template A using
ionic liquid 4d, DMSO, and betaine as enhancing reagents (70 mM each).
The protocol of ‘‘slowdown’’ PCR was employed for DNA amplification
(ESI-1). Lane M, 100 bp DNA ladder; lane C, control (no ionic liquid,
KBr, DMSO, or betaine). DNA samples were analyzed by 1% agarose
gel and visualized using ethidium bromide staining.
Fig. 4 Screening of ionic liquids (70 mM each) used to promote PCR
amplification of a normal-GC (55% GC content), 501 bp gene fragment B.
c
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methodology to find use in amplifying difficult genes, parti-
cularly for PCR systems that were previously shown to be
unsuccessful or unsatisfactory. This work provides a starting
point en route to a new class of enhancers for PCR ampli-
fication of DNA. We are currently investigating the general
applicability of this protocol for PCR amplification of a broad
range of genes and will report our results in due course.
We gratefully acknowledge support of this work by the
National Science Council of Taiwan, ROC (NSC-100-2113-
M-194-003-MY3 and NSC-99-2811-M-194-027) and the
Advanced Institute for Manufacturing with High-Tech
Innovations (AIM-HI at CCU). Y.S. thanks NSC of Taiwan
for a Research Postdoctoral Fellowship. We also thank
reviewers for valuable and constructive comments.
Fig. 5 PCR amplification of normal GC 501 bp DNA template B using
ionic liquid 4d, DMSO, and betaine as enhancing reagents (70 mM each).
Lane M, 100 bp DNA ladder; lane C, control (no ionic liquid, KBr,
DMSO, or betaine). DNA samples were analyzed by 1% agarose gel
and visualized using ethidium bromide staining.
Notes and references
1 For recent reviews on ionic liquids, see: (a) S. Sowmiah, C. I. Cheng
and Y.-H. Chu, Curr. Org. Synth., 2012, 9, 74; (b) R. Giernoth,
Angew. Chem., Int. Ed., 2010, 49, 2834; (c) D. Coleman and
N. Gathergood, Chem. Soc. Rev., 2010, 39, 600; (d) S. Sowmiah,
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14, 3780; (e) N. V. Plechkova and K. R. Seddon, Chem. Soc. Rev.,
2008, 37, 123.
2 (a) M.-C. Tseng, H.-T. Cheng, M.-J. Shen and Y.-H. Chu,
Org. Lett., 2011, 13, 4434; (b) C.-W. Chen, M.-C. Tseng, S.-K. Hsiao,
W.-H. Chen and Y.-H. Chu, Org. Biomol. Chem., 2011, 9, 4188;
(c) M.-C. Tseng and Y.-H. Chu, Chem. Commun., 2010, 46, 2983;
(d) M.-C. Tseng, M.-J. Tseng and Y.-H. Chu, Chem. Commun., 2009,
7503; (e) M.-C. Tseng, H.-C. Kan and Y.-H. Chu, Tetrahedron Lett.,
2007, 48, 9085; (f) Y.-L. Lin, H.-C. Kan and Y.-H. Chu, Tetrahedron,
2007, 63, 10949; (g) H.-C. Kan, M.-C. Tseng and Y.-H. Chu,
Tetrahedron, 2007, 63, 1644; (h) J.-Y. Cheng and Y.-H. Chu,
Tetrahedron Lett., 2006, 47, 1575; (i) M.-C. Tseng, Y.-M. Liang
and Y.-H. Chu, Tetrahedron Lett., 2005, 46, 6131; (j) Y.-H. Yen and
Y.-H. Chu, Tetrahedron Lett., 2004, 45, 8137; (k) J.-C. Hsu,
Y.-H. Yen and Y.-H. Chu, Tetrahedron Lett., 2004, 45, 4673.
3 PCR amplification of GC-rich DNA templates is often hampered by
the formation secondary structures such as hairpins, resulting in the
occurrence of nonspecific bands in gel electrophoresis: U. H. Frey,
H. S. Bachmann, J. Peters and W. Siffert, Nat. Protoc., 2008, 3, 1312.
4 R. K. Saiki, D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi,
G. T. Horn, K. B. Mullis and H. A. Erlich, Science, 1988, 239, 487.
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Fig. 6 Fluorescence annealing curves for GC-rich DNA duplexes
(PCR product A, 266 bp) in the presence of ionic liquid 4d or DMSO
(70 mM each). The curves were obtained by differentiating the
fluorescence signal from SYBR Green I in the presence of DNA while
heating from 60 to 98 1C in increments of 0.2 1C using a realtime PCR
instrument (curves were vertically shifted for clarity). The peak positions
represent the annealing temperatures Tm, written above each curve.
Tm values follow the trend 4d o DMSO B control.
experimental conditions (Tm = 93.9 1C). This fluorescence
annealing curve measurements confirmed that ionic liquid 4d
decreases stability of double-stranded DNA by lowering its
Tm value. This ionic liquid also effectively lowered the Tm of
normal-GC PCR adduct B: 82.2 and 85.9 1C with and without 4d,
and 85.7 1C with DMSO, respectively (Fig. S11, ESI-1w).
We reasoned that the organization of water molecules
around the bases in DNA should influence the duplex stability.
The addition of ionic liquids,9 which are less polar than water,
is expected to reduce the dielectric constant of the solvent,
and weaken interstrand interactions, which thereby leads to
decreased rigidity, and ultimately destabilizes and relaxes the
DNA duplexes. Despite our experimental results that show
that ionic liquids lower annealing temperatures and promote
PCR amplification of DNA, the exact functions of ionic
liquids remain enigmatic and, without doubt, more work is
needed.
In conclusion, we have examined twenty four ionic liquids
as potential PCR enhancing reagents, compared with known
enhancers governing effective PCR amplification of both
normal- and high-GC DNA, and identified ionic liquids with
a set of optimized conditions that allow successful PCR
amplification of GC-rich DNA under low ionic liquid concen-
tration and normal-GC DNA at substantially lower temperatures.
To our knowledge, this is the first report on ionic liquid enhance-
ment in gene amplification. More broadly, we expect this
7 P. Verhasselt, F. Poncelet, K. Vits, A. Van Gool and J. Vanderleyden,
FEMS Microbiol. Lett., 1989, 59, 135.
8 In the literature, DMSO at 5–10% (v/v) concentrations (i.e., 0.7–1.4 M)
is best known to improve PCR amplification of GC-rich DNA
sequences6.
9 C. Reichardt, Green Chem., 2005, 7, 339.
c
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Chem. Commun., 2012, 48, 5325–5327 5327