77

Construction of Synthetic Genes by Polymerase Chain Reaction

Patrick J. Dillon and Craig A. Rosen

1. Introduction Although the polymerase chain reaction (PCR) (1,2) is invaluable for the cloning and manipulation of existing DNA sequences, PCR also makes it possible to create new DNA fragments consisting of a nucleic acid sequence that is specified entirely by the investigator. In this chapter we describe a simple two-step PCR method for the rapid construction of synthetic genes (3). This method is based on early observations by Mullis et al. (4) in which multiple overlapping oligonucleotides could be used to generate synthetic DNA through several sequential rounds of Klenow based PCR amplification. The method described in this chapter utilizes the thermostable Taq polymerase and allows for the generation of synthetic genes in as little as 1 d. This method has proven useful in studies in which synthetic genes were constructed for the HIV-2 Rev protein (3,5) and the Wilms' tumor locus zinc finger protein (6). Furthermore, this method has been successfully employed in extensive mutagenesis of the HIV-1 rev response element (7). Examples for the use of designing synthetic genes include (l) the generation of unique chimeric constructs to study structure-function relationships and domain-swapping effects for a variety of related and unrelated proteins; (2) large-scale alterations or mutational analysis of motifs presenting either proteins or transcriptional elements (e.g., promoters, terminators, and so forth); (3) the creation of unique or novel promoters or proteins; and (4) saturation mutagenesis of genes through the use of random nucleotide incorporation or the use of deoxyinosine in the design of the gene sequence. The principles of this two-step PCR method for the construction of synthetic genes are outlined in Fig. 1. In this method, two sequential PCR reactions are used, the first PCR reaction generates a template DNA corresponding to the synthetic gene, which is then amplified in a second PCR reaction. Before starting this procedure, the investigator must design the construct and determine the nucleic acid sequence of the desired synthetic gene. Once this has been accomplished, oligonucleotides that span the length of the gene must be designed and synthesized. In general, an even number of oligonucleotides should be synthesized and should contain overlaps that are between 15 and 30 nt long. The orientation of the oligonucleotides should be similar to that in panel A
From: The Nucleic Acid Protocols Handbook Edited by: R. Rapley Humana Press Inc., Totowa, NJ

609

610

Dillon and Rosen

A
2

SYNTHETIC GENE SEQUENCE

4

1JtJ
2

3~ First Cycle 4--4~-

....... nr~

2'

-<.IV .... II(E---- Second Cycle

3' 4

Third Cycle

C
I

SYNTHETIC GENE SEQUENCE

~
Fig. 1. Description of two-step PCR method for construction of synthetic genes. (A) Schematic of design and orientation of overlapping oligonucleotides for first PCR reaction. (B) Diagram of oligonucleotide extensions during initial cycle of first PCR. (C) Schematic of design and orientation of flanking primers used in the second PCR reaction.

of Fig. 1. It is imperative that the outermost oligonucleotides correspond to opposite strands and be positioned so that they will extend inward toward each other over the gene. The number and length of individual oligonucleotides will vary according to the size of the synthetic DNA to be generated. Typically, oligonucleotides should be between 60 and 125 nt long. For example, four oligonucleotides can be used to synthesize a 325-bp DNA, whereas eight oligonucleotides can be used to generate a 765-bp construct. For this method, crude oligonucleotide preparations are used and it is not necessary for any additional purification of the oligonucleotides. Once the oligonucleotides have been obtained, the first step of the method is to mix the overlapping oligonucleotides in a standard PCR reaction. Panel B in Fig. 1 shows how four overlapping oligonucleotides would be extended through the first few cycles of PCR. The first PCR should be carried out with enough cycles to generate a doublestranded PCR product that spans the full length of the synthetic gene. The second step in the method is to take a small aliquot of the first peR reaction and amplify the syn-

Construction of Synthetic Genes

611

thetic gene in a second-strand peR reaction that contains short flanking primers (A,B) as illustrated in panel C. The sequence of the flanking primers should contain restriction sites to facilitate cloning. This procedure provides ample amounts of DNA for subsequent cloning into appropriate vectors. This method is an extremely powerful tool for the manipulation of nucleic acid and construction of synthetic genes.
2. Materials
1. lOX PCR buffer: 500 roM KCl, 100 roM Tris-HCl, pH 8.0, 15 roM MgCl z

2. 3. 4. 5. 6.

lOX dNTP solution: 2 roM each dATP, dCTP, dGTP, and dTTP. Taq DNA polymerase. Sterile water. Sterile mineral oil. Overlapping oligonucleotides that span the length of the DNA segment to be synthesized (see Note 1). An even number of oligonucleotides should be used and contain overlaps of at least 15 nt (see Note 2). 7. Flanking oligonucleotide primers that contain suitable restriction sites for cloning. 8. Agarose gel for analysis of PCR products (see Chapter 13).

3. Methods
1. Set up the first PCR reaction as follows: a. 1O!JL of lOX PCR buffer b. 1O!JL of lOX dNTP solution c. 0.5 J..lg each of overlapping oligonucleotides d. 2.5 U of Taq DNA polymerase; e. Sterile water to a final volume of 100 !JL f. Overlay sample with 50 !JL of sterile mineral oil 2. Amplify by PCR using the following cycle profile (see Notes 3, 4, and 5): a. Initial denaturation 94C, 5 min b. 10 main cycles 94C, 1 min (denaturation) 55C, 1 min (annealing) noc, 1 min (extension) c. Final extension noc, 5 min 3. Set up second PCR reaction as follows: a. 10 of lOX PCR buffer b. 10!JL of lOX dNTP solution c. 1!JL of first PCR reaction as template d. 1 J..lg of each flanking primer e. 2.5 U Taq polymerase f. Sterile water to final volume of 100 !JL g. Overlay sample with 50 !JL of sterile mineral oil 4. Run the second PCR using the following cycle profile: a. Initial denaturation 94C, 5 min b. 25 main cycles 55C, 1 min noc, 1 min 94C, 1 min c. Final extension noc, 5 min 5. Analyze 10 !JL of the first and second PCR reactions by agarose gel electrophoresis (see Chapter 13). A faint smear should be present in the first PCR reaction, and a band corresponding to the size of the desired product should be present in the second PCR reaction (see Note 6).

612

Dillon and Rosen

6. Digest the product from the second PCR reaction and clone into a suitable vector (see Note 7).

4. Notes
1. It is not necessary to purify oligonucleotides when using this method. In addition, there is no need to phosphorylate the oligonucleotides, as no ligation steps are used in this protocol. 2. Although it is suggested that an even number of overlapping oligonucleotides be used, an odd number may be used as long as the outermost oligonucleotides are on opposite strands and will extend inward toward each other. 3. The number of cycles needed for the first PCR reaction can be varied depending on the number of oligonucleotides used. In theory, only three cycles should be necessary for fulllength template synthesis using four oligonucleotides, whereas four cycles would be necessary if eight oligonucleotides were used. 4. The flanking primers should not be included in the first PCR reaction, as their addition results in the generation of many different-sized products that do not amplify well in the second PCR reaction. 5. It should be noted that the nucleic acid sequences of the overlaps may influence the annealing temperatures used during the first PCR reaction. 6. This method has been successful for the generation of synthetic constructs over 750 bp long. 7. When using this protocol for generating synthetic constructs, it is advisable to sequence the final product to assure that the sequence is correct. The error rate for this method should approximate that observed for other PCR protocols using Taq polymerase.

References
1. Saiki, R. K., Gelfand, D. H., Stofel, S., Scharf, S. J., Higuchi, R., Hom, G. T., Mullis, K. B., and Ehrlich, H. A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. 2. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Hom, G. T., Ehlrich, H. A., and Amheim, N. (1985) Enzymatic amplification of P-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350-1354. 3. Dillon, P. J. and Rosen, C. A. (1990) A rapid method for the construction of synthetic genes using the polymerase chain reaction. BioTechniques 9, 298-299. 4. Mullis, K., Faloona, F., Scharf, S., Saiki, R., Hom, G., and Erlich, H. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. BioI. 51, 263-273. 5. Dillon, P. J., Nelbock, P., Perkins, A., and Rosen, C. A. (1990) Function of the human immunodeficiency virus types I and 2 Rev proteins is dependent upon their ability to interact with a structural region present in the env gene mRNA. J. Virol. 64,4428-4437. 6. Rauscher III, F. J:, Morris, J. F., Joumay, O. E., Cook, D. M., and Cuffan, T. (1990) Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence. Science 250,1259-1262. 7. Olsen, H. S., Beidas, S., Dillon, P. J., Rosen, C. A., and Cochrane, A. W. (1991) Mutational analysis of the HIV-1 Rev protein and its target sequence, the rev response element. J. Acquired Immun. Defic. Syndrome 4, 558-567.