Journal of Biotechnology 137 (2008) 1–7

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

A high-throughput and single-tube recombination of crude PCR products
using a DNA polymerase inhibitor and type IIS restriction enzyme
Ippei Kotera a , Takeharu Nagai b,∗ab

PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan
Laboratory for Nanosystems Physiology, Research Institute for Electronic Science, Hokkaido University, Kita-20 Nishi-10 Kita-ku, Sapporo, Hokkaido 001-0020, Japana r t i c l e

i n f oArticle history:
Received 31 March 2008
Received in revised form 22 June 2008
Accepted 7 July 2008
Keywords:
DNA manipulations
DNA recombination
DNA cloning
Type IIS restriction endonucleaseHigh-throughput

a b s t r a c t
Type IIS restriction enzymes have been successfully used as “universal” restriction enzymes in DNA
manipulations. We took a step further to develop a rapid technique for recombining DNA fragments, fully
automatic single-tube recombination (FASTR), which enables multiple-fragment DNA recombination in asingle step. Crude PCR products are directly mixed with both type IIS restriction endonuclease and DNA
ligase to initiate a spontaneous and one-way recombination reaction. Highly efﬁcient DNA recombination
can be achieved by an inhibition of DNA polymerase with aphidicolin and a selective digestion of template
DNAs by DpnI, a restriction enzyme to digest hemi-methylated DNA in the reaction solution; thereby theentire procedure takes less than 15 min. Owing to its simplicity, efﬁciency and rapidity, one-step FASTR canbe applied to a wide range of DNA manipulations including those involving high-throughput applications
where signiﬁcant reduction in time and cost is expected.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction
There have been various approaches to making cloning simpler
or faster: instead of using classical restriction enzymes and DNA
ligase, DNA recombination systems from different organisms havebeen used to manipulate plasmid DNA (Hartley et al., 2000; Liuet al., 1998). These techniques have made some repetitious geneclonings easier, but because they are based on DNA recombination,
special sequences must accompany the gene to be cloned and thedestination vector. These sequences are not removed during therecombination reaction, which is problematic when one tries tofuse one gene to another with a deﬁned linker sequence. Anotherproblem is that if a destination vector is not available for a certain
application, one has to make it through conventional plasmid construction. Thus, recombination-based cloning techniques can lackto some degree the ﬂexibility of classical cloning technology.
Type IIS restriction enzymes digest DNA sequence outside of
their recognition sequence (Kleid et al., 1976; Takemori et al., 2002).
With recognition sequences on primers, type IIS enzymes can beutilized as a universal restriction enzyme for the cloning of PCR
products (Podhajska and Szybalski, 1985). We refocused on thisclassic enzyme because of its versatility and potential for a fast
spontaneous DNA recombination reaction. With an inhibition of∗ Corresponding author. Tel.: +81 11 706 3833; fax: +81 11 706 4968.
E-mail address: tnagai@es.hokudai.ac.jp (T. Nagai).
0168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2008.07.1816

residual thermostable DNA polymerase activity and selective digestion of template DNAs, we are now able to recombine multiple DNA
fragments directly from crude PCR solution in a single tube within
15 min.
2. Materials and methods
2.1. FASTR reaction
PCR was carried out using a high-ﬁdelity thermostable DNApolymerase, KOD-plus (Toyobo, Osaka, Japan) according to the
manufacturer’s protocol. Brieﬂy, the reaction buffer contained
1× KOD-plus buffer, 2 mM dNTPs, 25 mM MgSO4 , 1 unit of KODplus, 25 pmol each of forward/reverse primers, and 50 ng eachof template plasmids, in a total volume of 50 ␮l. The templateplasmids for mSECFP (Matsuda et al., 2008) and mVenus (Venus(Nagai et al., 2002) with A206K mutations (Zacharias et al., 2002))
were in a pcDNA3.0 vector (Invitrogen, CA, USA), and mCherry(Shu et al., 2006) was in a plant expression vector. pRSET B vector(Invitrogen, CA, USA) was modiﬁed by site-directed mutagenesis todelete an LguI site at nucleotide position 2830. The PCR conditions
were as follows: 94 ◦ C/2 min (94 ◦ C/15 s, 60 ◦ C/30 s, 68 ◦ C/90 s), 35
cycles, 68 ◦ C/5 min. One microliter of the PCR solution from eachsample was transferred to a mixture containing the following
reagents in a total volume of 20 ␮l: 17 mM Tris–acetate, 25 mM
Tris–HCl (pH 7.5), 5 mM MgCl2 , 5 mM magnesium acetate, 33 mM
potassium acetate, 5 mM ATP, 5 mM DTT, 63 ␮g/ml BSA, 5 U of LguI

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I. Kotera, T. Nagai / Journal of Biotechnology 137 (2008) 1–7(Fermentas, ON, Canada), 400 U of T4 DNA ligase (NEB, MA, USA),
20 U of DpnI (NEB, MA, USA), and 5 mM aphidicolin (Calbiochem,
CA, USA). The mixture was incubated without agitation at room
temperature for various lengths of time. After incubation, 5 ␮l ofthe mixture was used to transform Escherichia coli (JM109 DE3,
Promega, WI, USA). The sequences of the primers used in the
FASTR reaction were as follows: pRSET forward primer: GCTACTGCTCTTCGTGCGCTGGTACCATGGAATTCGAA, pRSET reverse primer:
CTGATAGCTCTTCTCACGTACAGATCCCGACCCATTTG, GFP-variants
forwardprimer:GCTAGCTCTTCAGTGATGGTGAGCAAGGGCGAand GFP-variants reverse primer: CTAGGCTCTTCTGCACTTGTACAGCTCGTCCATGC. For multiple-fragment FASTR the following
sets of primers were used to amplify the inserts: GFP-variants forward 1: GCTAGCTCTTCAGTGATGGTGAGCAAGGGCGA, GFP-variants
reverse 1: CTAGGCTCTTCTACCCTTGTACAGCTCGTCCATGC, GFPvariants forward 2: GCTAGCTCTTCAGGTATGGTGAGCAAGGGCGA,
GFP-variants reverse 2: CTAGGCTCTTCTGCACTTGTACAGCTCGTCCATGC.
2.2. Fluorescent colony imaging
For the single-color imaging of mSECFP-expressed E. coli
colonies, an LAS-1000plus image analyzer (Fujiﬁlm, Tokyo, Japan)
was used to take ﬂuorescent images. For multicolor imaging, a
hand-made device equipped with a Mercury lamp, interference ﬁlters, optical ﬁber cables, and video CCD camera (WAT-120N, Watec,
Yamagata, Japan) was used. The ﬁlter combination was as follows:
440/21 excitation and 480/30 emission for mSECFP, 500/24 excitation and 535/26 emission for mVenus and 540/30 excitation and575LP for mCherry. All images were acquired using ImageJ software. MetaMorph (Molecular Devices, CA, USA) was used for image
analysis.
2.3. Determination of FRET efﬁciency
Venus/T-sapphire fusion protein was mixed with 0.05%
trypsin–EDTA (Gibco, CA, USA) in PBS and incubated at 37 ◦ C for
1 h. The samples with and without trypsin were analyzed by F-2500
ﬂuorescence spectrophotometer (Hitachi, Tokyo, Japan). The peakvalues of the donor protein (T-sapphire) before and after the trypsin
treatment were used to calculate the FRET efﬁciency, according to
the following equation:
FDA
E =1−
FA
where E is the FRET efﬁciency of the donor/acceptor pair, FDA is theﬂuorescence intensity of the donor in the presence of the acceptor(before trypsin treatment), and FA is the ﬂuorescence intensity ofthe donor in the absence of the acceptor (after trypsin treatment).
3. Results
3.1. The concept of the FASTR reaction
The DNA cleavage site of type IIS endonucleases is at a distancefrom their DNA recognition site, thus these enzymes create DNA
overhangs of any nucleotide sequence (Szybalski et al., 1991). Thisfeature is advantageous for DNA manipulation in many ways. In
carefully designed plasmid constructions, the DNA recognition site
for type IIS enzymes can be removed, allowing the ligated ﬁnal
product to be tolerant of the endonuclease that created the overhangs (Lebedenko et al., 1991). This method is more ﬂexible thanthose using conventional type II endonucleases, because the joint

sequence can be designed freely. Another advantage is that thecleavage site does not have to be palindromic, thus preventing
undesired homo-ligation between inserts or vectors. We reasoned
that, with these features, it might be possible to have both ligation and endonuclease digestion occur simultaneously in the sametube.
As shown in Fig. 1A, all the terminals of the DNA fragments in
FASTR carry cleavage sites for type IIS endonuclease followed byrecognition sites for the endonuclease. The recognition sites arealways outside of the cleavage sites, so after digestion by type IIS
endonuclease, no recognition site remains on the DNA fragment.
Because both the endonuclease and the ligase are present in thereaction mixture, equilibrium between digestion and ligation isachieved, as long as the ligated product carries the recognition site
for the endonuclease. Once ligation between the designated DNA
fragments is completed, there is no more digestion even in the presence of the endonuclease, because of the loss of the recognition site.
It is this directional reaction that drives the designated products outof the equilibrium, enabling the recombination reaction in a single
step (Fig. 1C). The sequence of the cleavage site is independent of
the recognition site, allowing the terminus of each DNA fragment tohave a speciﬁc sticky end for a speciﬁc partner fragment. If a threenucleotide sticky end is used for FASTR, one can theoretically use
43 , or 64, speciﬁc sticky ends, enabling multiple-fragment ligations(Fig. 1B).
3.2. FASTR reaction using gel-puriﬁed DNA fragments
To demonstrate this single-step recombination reaction, we
ampliﬁed both the insert and vector fragments by PCR using
pairs of primers with speciﬁc cleavage and recognition sites oneach end. The ampliﬁed fragments were gel-extracted and mixed
with type IIS endonuclease (LguI) and T4 DNA ligase. After 2 hof incubation at room temperature, 5 ␮l of the reaction mixture was used directly to transform chemically competent E. coli.
To our surprise, eight samples from eight colonies gave positive
results for both single- and double-fragment insertions (Fig. 2A).
DNA sequencing conﬁrmed that the sequences of the joint region
were exactly as designed, verifying the validity of the FASTR
reaction (Fig. 2B).
3.3. FASTR reaction using crude PCR products
Encouraged by the result, we thought it might be possible torecombine crude PCR products in a single step. In our early attempts
to use this single-tube method, only few positive colonies wereobtained (data not shown). We reasoned that the residual activityof the thermostable DNA polymerase would convert the sticky ends
to blunt ends, giving rise to self-ligation of the vector plasmid. If so,
a DNA polymerase inhibitor might help to reduce the unwanted
self-ligation.
Aphidicolin has been known to inhibit the alpha subtype of
DNA polymerase in eukaryotes, as well as some types of archaeal
DNA polymerases (Ikegami et al., 1978; Klimczak et al., 1985). The
KOD-plus DNA polymerase was derived from archaeal Pyrococcus kodakaraensis (Takagi et al., 1997). We therefore hypothesizedthat aphidicolin could inhibit the residual activity of the KODplus DNA polymerase present in the PCR product solution, thus
allowing a higher recombination rate in the following FASTR reaction. To test the hypothesis, aphidicolin at a ﬁnal concentration of
2.5 mM was added to the PCR product solution, and 1 ␮l each of
the insert (cyan ﬂuorescent protein, mSECFP) and vector (pRSET
B) with the LguI site mutated) solutions were mixed with LguI
restriction endonuclease and T4 DNA ligase. DpnI was also includedin the reaction to digest the residual template DNA. After vari-

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3

Fig. 1. Schematic representation of the FASTR reactions. (A) PCR products (top and bottom) are treated with both type IIS endonuclease and DNA ligase to induce equilibrium
reactions of digestion and ligation. Once the FASTR product is assembled (middle), the reaction stops, because the ﬁnal product lacks the recognition site for the endonuclease.
Blue boxes represent the recognition site for type IIS endonuclease and red lines are the cleavage site of the endonuclease. Vector DNA is in orange and insert DNA is in green.
(B) An example of FASTR product with multiple inserts. The inserts are indicated in green and purple. (C) Outline of the FASTR protocol. The entire procedure of FASTR iscarried out in a single tube and takes less than 15 min.

ous incubation times at room temperature, JM109 DE3 competentcells were transformed with 5 ␮l of the FASTR reactant. With
15 min of FASTR reaction at room temperature, more than half ofthe colonies were ﬂuorescent upon illumination of the plate by
470 nm blue LED. The recombination efﬁciency reached close to
80% when the incubation time was extended to 2 h (Fig. 2C andD).

3.4. Multiple DNA fragment assembly by FASTR reactions
We next examined the possibility of performing multiplefragment FASTR with the crude PCR products. The procedures
were essentially the same as for single-fragment FASTR, exceptthat the joint sequences were designed so that the insertion
fragments would be fused in the correct direction and order4

I. Kotera, T. Nagai / Journal of Biotechnology 137 (2008) 1–7

Fig. 2. High recombination rate of FASTR. E. coli colonies were obtained by the transformation of FASTR products. The colonies were subjected to colony PCR for insert
analysis. (A) Eight randomly picked colonies were positive for single and double insertions. (B) The joints between the vector and insert were DNA sequenced to conﬁrm thevalidity of the method. The FASTR reaction was carried out for various lengths of time. The number of transformed colonies reached near-plateau at 60 min (C); however,
the recombination rate changed little after 15 min (D). Black bars represent the total number of colonies, and gray bars represent the number of ﬂuorescent colonies. The
recombination rate was calculated by dividing the number of ﬂuorescent colonies by the total number of colonies.

(Fig. 1B). We ampliﬁed the insert fragments of cyan ﬂuorescent protein (mSECFP), yellow ﬂuorescent protein (mVenus), andred ﬂuorescent protein (mCherry) along with the vector fragment of pRSET B. The fragments were then mixed to assemble
pRSET–mSECFP–mVenus and pRSET–mSECFP–mVenus–mCherry
constructs. The FASTR reaction was performed at room temperature for 1 h. We conﬁrmed that close to half of the ﬂuorescentcolonies were double positive for mSECFP and mVenus ﬂuorescence in pRSET–mSECFP–mVenus construct, and around 20% weretriple positive for mSECFP, mVenus, and mCherry ﬂuorescence inpRSET–mSECFP–mVenus–mCherry sample (Table 1 and Fig. 3A).
These colonies were further analyzed by colony PCR to conﬁrm theinserts (Fig. 3B).

3.5. High-throughput screening of a high-performance FRET
probe by FASTR
To demonstrate the feasibility of FASTR in a practical application, we constructed a GFP-based Förster resonance energy transfer

Table 1

InsertNumber of
positive colonies

Number of
ﬂuorescent colonies

Recombination
rate (%)

CFP+Venus
CFP+Venus+mCherry

517
50

1050
242

49.24
20.66

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5

Fig. 3. Double and triple insertion by FASTR. (A) Fluorescent images of colonies expressing mSECFP, Venus, and/or mCherry were taken with multiple ﬁlter sets. The images
from different color channels were overlaid and pseudocolored. Yellow colonies in the double insertion and white colonies in the triple insertion images are double and triple
positives, respectively. (B) Insert lengths of the positive colonies selected by ﬂuorescence were conﬁrmed by colony PCR.

(FRET) pair with various linker regions to maximize FRET efﬁciency. FRET-based GFP probes have been widely used in biologicalstudies as a genetically encoded molecular sensor (Piston and Kremers, 2007). One obstacle to the technology is that FRET efﬁciencychanges dramatically with very small changes in the distance andrelative orientation between ﬂuorophores (Lakowicz, 2006). The
sensitivity of a FRET pair is so acute that even a single amino-acidinsertion or deletion in the linker region is enough to affect the FRET
efﬁciency. Thus, the construction of a FRET pair requires laborious
linker optimization by trial and error (Nagai and Miyawaki, 2004).
Here, we used FASTR to fuse a UV-excitable green ﬂuorescent protein (T-Sapphire) (Zapata-Hommer and Griesbeck, 2003) toVenus, with various linker lengths between them (Fig. 4A). Thirteen
reverse primers were designed for C-terminal deletion mutants ofVenus, and seven forward primers for N-terminal T-Sapphire deletions. A total of 20 deletion mutants were PCR-ampliﬁed. Then 1 ␮lof the crude PCR products from each sample were mixed in one
tube along with FASTR reagents. The FASTR reaction should recombine two inserts and a vector with correct order but in random
combinations, giving rise to a total of 91 mutant combinations.
After 2 h of incubation at room temperature, JM109 competentcells were transformed with 10 ␮l of the reactant. E. coli plates
were screened for yellowish ﬂuorescent colonies using a 405 nm
light source. DNA sequencing of the linker region and spectroscopic
analysis of the expressed proteins identiﬁed the clones with high
FRET efﬁciency (Fig. 4B). The FRET efﬁciency of the best clone we
obtained was determined to be 67% by cleaving the linker region(Fig. 4C), demonstrating that FASTR is an ideal method for creating alarge number of multiple-fragment constructs for high-throughput
screening.

4. Discussion
Although cloning techniques with type IIS restriction endonucleases and DNA ligases have been widely used, the endonucleasemediated single-step recombination in our method is much fasterthan previous approaches. As far as we know, this is the ﬁrst
report which describes the spontaneous and directional singletube recombination with a type IIS restriction endonuclease andDNA ligase. The reaction allows highly efﬁcient DNA recombination, while keeping a high ﬂexibility of class IIS restriction enzymes.
Another advantage of our approach is the ability to directly recombine crude PCR products. This was made possible by adding anarcheal DNA polymerase inhibitor to the FASTR reaction. Although
we have not tried a monoclonal antibody against the polymerase, it
might also be used to inactivate the residual polymerase activity, as
it is for hot-start PCR (Kellogg et al., 1994). DpnI was also included in
the reaction to selectively digest the bacterially expressed template
DNA in the crude PCR products.
We primarily focused our efforts on improving the rapidityof the method, because we thought that raising the speed limit
for DNA recombination would enhance research in many ﬁelds,
especially ones involving high-throughput analysis. One such ﬁeld
is the development of GFP-based FRET probes, which are now
indispensable in the biological sciences. A typical FRET probe
has ﬂuorescent donor and acceptor domains along with a sensor domain to detect biological signals. High FRET efﬁciency isachieved only when the distance between ﬂuorophores is comparable to the Förster distance, and the relative dipole moments of
the ﬂuorophores are parallel to each other. The distance between
ﬂuorophores is largely affected by the linker sequences between

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I. Kotera, T. Nagai / Journal of Biotechnology 137 (2008) 1–7

Fig. 4. Optimization of a GFP-based FRET pair. (A) A schematic representation of FRET pair optimization by FASTR. GFP variants with various lengths of linker regions are
PCR-ampliﬁed (top). FASTR intermediates are indicated in the middle panel. The ﬁnal products are represented in the bottom panel. An N-terminal deletion is indicated as
N1 and a C-terminal deletion as C1. ‘n’ deletions are represented as Nn or Cn. (B) Fluorescence spectra of Venus (511 nm) and T-Sapphire (527 nm) fusion proteins
with various linker regions. The spectra were obtained by 395 nm illumination and ﬂuorescence scanning with a 5 nm slit. The curves were normalized to the ﬂuorescenceintensity at 530 nm. (C) Venus-T-sapphire (C2–N0) fusion protein was cleaved into separate ﬂuorophores by trypsin. The FRET efﬁciency of the pair was calculated by
ﬂuorescence recovery of the donor protein (T-sapphire).

the domains, and the relative orientation of the ﬂuorophores canbe changed by using circular permutations of the ﬂuorescentproteins (Nagai et al., 2004). Since it is very difﬁcult to predict
the structural state of the ﬂuorophores by calculation, one has
to optimize these parameters through empirical methods. FASTR
technology can contribute to the development of such probes,
because it can assemble multiple domains of FRET probes simultaneously, saving the time and cost of constructing numerous

combinations of linkers and domains to obtain a favorable FRET
signal.
A possible drawback of FASTR methodology would be therequirement of primers for each FASTR reaction. Although thecost and availability of synthetic oligonucleotides have been
greatly improved, the requirement might be an obstacle to someresearchers. Another drawback would be the imcompatibility of
FASTR products with conventional restriction enzymes; the liga-

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tion site of the FASTR products cannot be recut with conventional
restriction enzymes. We recommend another round of FASTR reaction for an additional plasmid modiﬁcation, and colony PCR for
insert check.
FASTR technology is among the quickest in the latest generationof recombinant technologies, while it achieves the high efﬁciencyof recombination-based methods and the ﬂexibility of conventional cloning. These advantages should be applicable to most DNA
manipulation procedures, including high-throughput DNA manipulations, where signiﬁcant savings in time and cost are expected.
Acknowledgments
We are thankful to R.Y. Tsien for the gift of mCherry plasmid.
We also thank Wataru Tomosugi for the construction of T-Sapphire
plasmid, by mutating EGFP plasmid according to the publisheddata. This work was partly supported by grants from ScientiﬁcResearch on Advanced Medical Technology of the Labor, Health andWelfare of Japan to T.N., the Japanese Ministry of Education, Scienceand Technology to T.N., and Precursory Research for Embryonic Science and Technology (PRESTO) of the Japan Science and Technology
Agency (JST) to I.K.
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