Analytical Biochemistry 415 (2011) 21–26 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio Quikgene: A gene synthesis method integrated with ligation-free cloning Yanjun Mao a,1, Juanyu Lin a,1, Aibin Zhou a, Kunmei Ji b, Jennifer S. Downey c, Ruichuan Chen a, ⇑ Aidong Han a, a MOE Key Laboratory for Cell Biology, School of Life Sciences, Xiamen University, Fujian 361005, China b School of Medicine, Shenzhen University, Shenzhen, Guangdong 518060, China c Division of Biomedical Science, Herman Ostrow School of Dentistry of University of Southern California, Los Angeles, CA 90089, USA article info abstract Article history: Gene synthesis is a convenient tool that is widely used to make genes for a variety of purposes. All current Received 17 November 2010 protocols essentially take inside-out approaches to assemble complete genes using DNA oligonucleotides Received in revised form 30 March 2011 or intermediate fragments. Here we present an efficient method that integrates gene synthesis and clon- Accepted 4 April 2011 ing into one step. Our method, which is evolved from QuikChange mutagenesis, can modify, extend, or Available online 15 April 2011 even de novo synthesize relatively large genes. The genes are inserted directly into vectors without liga- tions or subcloning. We de novo synthesized a 600-bp gene through multiple steps of polymerase chain Keywords: reaction (PCR) directly into a bacterial expression vector. This outside-in gene synthesis method is called Gene synthesis Quikgene. Furthermore, we have defined an overlap region of a minimum of nine nucleotides in insertion Ligation-free cloning QuikChange primers that is sufficient enough to circularize PCR products for efficient transformation, allowing one to Quikgene significantly reduce the lengths of primers. Taken together, our protocol greatly extends the current length limit for QuikChange insertion. More importantly, it combines gene synthesis and cloning into one step. It has potential applications for high-throughput structural genomics. Ó 2011 Elsevier Inc. All rights reserved. Gene synthesis provides convenient access to genes that are of genomic studies. Currently, several computer programs can help to interest for a multitude of studies. With genomic sequences of design genes into the sequences appropriate for target hosts [4]. many species publicly available, one can theoretically obtain any Gene synthesis can be carried out by chemical synthesis, ligase- gene by de novo synthesis. Standard methods involve extensive based synthesis, or polymerase chain reaction (PCR)2-based syn- time and expense after isolating or requesting complementary thesis. Khorana and coworkers first used chemical synthesis to gen- DNAs (cDNAs) and designing primers to clone the genes of interest erate yeast transfer RNA (tRNA) in 1972 [5,6]. A synthesized into various expression vectors. In addition, by expressing proteins promoter was found to be functional, and a gene coding for a func- outside of their origins, codon use can play vital roles in whether or tional protein, somatostatin, was successfully synthesized and ex- not there is any successful expression [1]. The negative factors, pressed in Escherichia coli [7,8]. However, the long DNA chemical including strong codon bias, unbalanced GC/AT composition, and synthesis was often discouraged due to the high cost and error rate unstable messenger RNA (mRNA), are often difficult to overcome [9]. Ligation and PCR extension were then developed using the short by traditional cloning methods. This is particularly true when using oligonucleotides that can be efficiently synthesized at a significantly bacterial or yeast expression systems for mammalian genes [2]. lower error rate [10,11]. These oligonucleotides were assembled into Furthermore, gene synthesis can often clean up those restriction complete genes by either ligation or PCR extension [12–14]. Liga- enzyme cleavage sites within genes for traditional cloning that re- tion-based oligonucleotide assembly requires those gel-purified lies on availability of cleavage sites [3]. QuikChange mutagenesis intermediate fragments and subsequent phosphorylation, which is (Stratagene) is often employed to remove those sites but can be time-consuming [12]. PCR-based oligonucleotide assembly can be tedious if there are too many. De novo gene synthesis can easily done in a single tube through dual asymmetrical PCR (DA–PCR) eliminate the majority of unwanted restriction sites by and a successive extension PCR protocol [9,14–17]. Young and Dong substitutions with alternative codons that are best used by appropriate expression hosts. Therefore, gene synthesis has be- come a preferable method to obtain genes of interest for structural 2 Abbreviations used: PCR, polymerase chain reaction; DA–PCR, dual asymmetrical PCR; Tm, melting temperature; TAE–PAGE, Tris–acetate–EDTA–polyacrylamide gel electrophoresis; IPTG, isopropyl b-d-1-thiogalactopyranoside; SDS, sodium dodecyl ⇑ Corresponding author. Fax: +86 0592 218 8173. sulfate; GST, glutathione S-transferase; DTT, dithiothreitol; aa, amino acids; E1A, early E-mail address: [email protected] (A. Han). region 1A; CR3, conserved region 3; SCAI, suppressor of cancer cell invasion; SRF, 1 These authors contributed equally to this work. serum response factor. 0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.04.004 22 Gene synthesis method integrated with cloning / Y. Mao et al. / Anal. Biochem. 415 (2011) 21–26 developed a combined protocol called two-step PCR using as short as products were analyzed using either 1.2% agarose or 5% native 25-nt oligonucleotides to make the syntheses error free [11]. In gen- Tris–acetate–EDTA–polyacrylamide gel electrophoresis (TAE– eral, PCR-assembled genes have very limited errors due to error cor- PAGE) gels. Reaction mixtures were cleaved by adding 1 llofDpnI rection activity of DNA polymerase that can be further enhanced by (New England Biolabs) for 2 h before further purification with the high-fidelity Taq DNA polymerases [18]. The error rate can also be CyclePure kit (Omega) and quantified at k260. PCR product (60 ng) further reduced by adding a mismatch-cleaning endonuclease was used to transform E. coli XL1 Blue competent cells, and trans- [17,19,20]. formants were selected on 2 Â YT agar plates (per liter: 10 g yeast Specific mutations, including substitution, deletion, and inser- extract, 16 g tryptone, 5 g NaCl, and 15 g agar) with the appropriate tion, are frequently required in structural and functional studies antibiotics according to a molecular cloning manual [24]. Colonies of genes. Single-stranded DNA and restriction ligation can be used were counted after overnight incubation at 37 °C. to generate desired mutations [21,22]. In comparison, QuikChange is a simpler and faster protocol that uses a pair of complete over- Small-scale expression tests lapping primers with melting temperatures (Tm values) over 78 °C according to the manufacturer’s protocol (Stratagene) [23]. Expression constructs were subsequently transferred into sev- The essential elements of QuikChange bipartite primers are two eral E. coli strains for expression, including regular BL21/DE3, overlapping regions complementary to opposite strands of the BL21/DE3 Gold, and BL21/DE3 Rosetta pLysS. Two individual colo- template. These primers are often 25–45 nt long; however, if a nies were inoculated, and colonies were grown in 3 ml of 2 Â YT li- large insertion is needed, the primers can be as long as 90 nt. The quid medium (per liter: 10 g yeast extract, 16 g tryptone, and 5 g length of primers is associated with not only the overall cost of NaCl) for approximately 6 h to reach an OD600 of 0.6. Bacterial cul- the mutagenesis experiments but also the error rate introduced ture (200 ll) was transferred into 800 ll of fresh 2 Â YT containing by oligonucleotide synthesis [11]. Therefore, similar to gene syn- 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) in 1.5-ml thesis, primer length should be kept to a minimum. tubes. These bacterial cultures were induced for 1 h with shaking QuikChange has proved to be a powerful mutagenesis tool. Here at different temperatures (37, 25, and 12 °C). The cells were col- we present a novel protocol, Quikgene, that uses the QuikChange lected by centrifugation at more than 10,000 rpm for 2 min. The li- insertion algorithm to assemble oligonucleotides into genes. How- quid medium was completely removed, and cell pellets were ever, unlike QuikChange, this method can assemble several hun- vortexed to homogeneity in 30 ll of distilled water and lysed with dreds of base pairs of genes. It can create complete de novo an equal volume of BugBuster (Invitrogen). At this point, the bacte- genes or just extend or modify existing genes. The final genes are rial mixtures were completely transparent unless the proteins directly synthesized on desired vectors without any ligation or were expressed in inclusion bodies. The mixtures as whole pro- subcloning steps. teins were analyzed on 15% sodium dodecyl sulfate (SDS)–PAGE. The supernatants after centrifugation (15,000g, >5 min, room tem- Materials and methods perature) can be used to check protein solubility. Expression vectors GST purification All vectors and plasmids are listed in Table 1. pXA90 is similar Expression strain BL21/DE3 Rosetta pLysS with an expression to pGEX-6p vector with two major differences: p15 replication ori- construct above was grown in 500 ml of 2 Â YT medium to OD600 gin and chloramphenicol resistance. pET-His was constructed from of 0.6 at 37 °C and induced with 0.25 mM IPTG overnight at pRSET with new multiple cloning and thrombin cleavage sites. All 37 °C. The cells were harvested and lysed by sonication in 40 ml plasmids were propagated in E. coli XL1 Blue and purified using the of buffer (50 mM Tris [pH 8.0], 500 mM NaCl, 10% glycerol, 3 mM Omega Miniprep kit. EDTA, 2 mM dithiothreitol [DTT], and 3 mM b-mercaptoethanol).