This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 5325–5327 5325

Cite this: Chem. Commun., 2012, 48, 5325–5327

Ionic liquids promote PCR amplification of DNAw

Yugang Shi,ab

Yen-Liang Liu,aPeng-Yeh Lai,

cMing-Chung Tseng,

aMin-Jen Tseng,

c

Yudong Liband 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.

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,

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

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.

aDepartment 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

bCollege of Food Science and Biotechnology, Zhejiang Provincial KeyLaboratory of Food Safety, Zhejiang Gongshang University,Hangzhou, Zhejiang 310035, PR China

c Institute of Molecular Biology, National Chung Cheng University,Minhsiung, Chiayi 62102, Taiwan, ROCw Electronic supplementary information (ESI) available: Fig. S1–S11and 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

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5326 Chem. Commun., 2012, 48, 5325–5327 This journal is c The Royal Society of Chemistry 2012

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).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

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.

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).

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

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.

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.

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This journal is c The Royal Society of Chemistry 2012 Chem. Commun., 2012, 48, 5325–5327 5327

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 1Cwith 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-GCDNA 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

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.

Notes and references

1 For recent reviews on ionic liquids, see: (a) S. Sowmiah, C. I. Chengand Y.-H. Chu, Curr. Org. Synth., 2012, 9, 74; (b) R. Giernoth,Angew. Chem., Int. Ed., 2010, 49, 2834; (c) D. Coleman andN. Gathergood, Chem. Soc. Rev., 2010, 39, 600; (d) S. Sowmiah,V. Srinivasadesikan, M.-C. Tseng and Y.-H. Chu, Molecules, 2009,14, 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. Liangand Y.-H. Chu, Tetrahedron Lett., 2005, 46, 6131; (j) Y.-H. Yen andY.-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 bythe formation secondary structures such as hairpins, resulting in theoccurrence 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.

5 (a) H. Hube, P. Reverdiau, S. Iochmann and Y. Gruel, Mol.Biotechnol., 2005, 31, 81; (b) J. P. Hapgood, J. Riedemann andS. D. Scherer, Cell Biol. Int., 2001, 25, 17; (c) S. Saccone,A. De Sario, G. Della Valle and G. Bernardi, Proc. Natl. Acad.Sci. U. S. A., 1992, 89, 4913.

6 (a) M. Ralser, R. Querfurth, H.-J. Warnatz, H. Lehrach,M.-L. Yaspo and S. Krobitsch, Biochem. Biophys. Res. Commun.,2006, 347, 747; (b) Z. Chen and Y. Zhang, Biochem. Biophys. Res.Commun., 2005, 333, 664; (c) M. Jung, J. M. Muche, A. Lukowsky,K. Jung and S. A. Loening, Anal. Biochem., 2001, 289, 292;(d) R. Chakrabarti and C. E. Schutt, Gene, 2001, 274, 293;(e) L. Le Cam, J. Polanowska, L. Fajas, E. Fabbrizio andC. Sardet, BioTechniques, 1999, 26, 840; (f) Q. Liu and S. S.Sommer, BioTechniques, 1998, 25, 1022; (g) W. Henke, K. Herdel,K. Jung, D. Schnorr and S. A. Loening, Nucleic Acids Res., 1997,25, 3957; (h) N. Baskaran, R. P. Kandpal, A. K. Bhargava, M. W.Glynn, A. Bale and S. M. Weissman, Genome Res., 1996, 6, 633;(i) M. K. Sidhu, M.-J. Liao and A. Rashidbaigi, BioTechniques,1996, 21, 44; (j) S. A. Filichkin and S. B. Gelvin, BioTechniques,1992, 12, 828; (k) D. Pomp and J. F. Medrano, BioTechniques, 1991,10, 58; (l) R. Bookstein, C.-C. Lai, H. To and W.-H. Lee, NucleicAcids Res., 1990, 18, 1666.

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 DNAsequences6.

9 C. Reichardt, Green Chem., 2005, 7, 339.

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.

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.

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