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Although the polymerase chain reaction (PCR) (1,2) is invaluable for the cloningand manipulation of existing DNA sequences, PCR also makes it possible to createnew DNA fragments consisting of a nucleic acid sequence that is specified entirely bythe investigator. In this chapter we describe a simple two-step PCR method for therapid construction of synthetic genes (3). This method is based on early observationsby Mullis et al. (4) in which multiple overlapping oligonucleotides could be used togenerate synthetic DNA through several sequential rounds of Klenow based PCRamplification. 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 methodhas proven useful in studies in which synthetic genes were constructed for the HIV-2Rev protein (3,5) and the Wilms' tumor locus zinc finger protein (6). Furthermore, thismethod has been successfully employed in extensive mutagenesis of the HIV-1 revresponse 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 alterationsor 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 randomnucleotide 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 genesare outlined in Fig. 1. In this method, two sequential PCR reactions are used, the firstPCR reaction generates a template DNA corresponding to the synthetic gene, which isthen amplified in a second PCR reaction. Before starting this procedure, the investigator must design the construct and determine the nucleic acid sequence of the desiredsynthetic gene. Once this has been accomplished, oligonucleotides that span the lengthof 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 and30 nt long. The orientation of the oligonucleotides should be similar to that in panel A

R.

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B 1JtJ 3 4 - - 4 ~ -n r

A

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 ofdesign and orientation of flanking primers used in the second PCR reaction.

of Fig. It is imperative that the outermost oligonucleotides correspond to oppositestrands and be positioned so that they will extend inward toward each other overthe 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 between60 and 125 nt long. For example, four oligonucleotides can be used to synthesize a325-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 showshow four overlapping oligonucleotides would be extended through the first few cyclesof 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 stepin the method is to take a small aliquot of the first peR reaction and amplify the syn-

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thetic gene in a second-strand 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 forsubsequent cloning into appropriate vectors. This method is an extremely powerfultool for the manipulation of nucleic acid and construction of synthetic genes.

lOX PCR buffer: 500 roM KCl, 100 roM Tris-HCl, pH 8.0, 15 roMMgClz2. lOX dNTP solution: 2 roM each dATP, dCTP, dGTP, and dTTP.3. Taq DNA polymerase.4. Sterile water.5. Sterile mineral oil.6. Overlapping oligonucleotides that span the length of the DNA segment to be synthesized

Note 1). An even number of oligonucleotides should be used and contain overlaps ofat least 15 nt Note 2).7. Flanking oligonucleotide primers that contain suitable restriction sites for cloning.

8. Agarose gel for analysis of PCR products Chapter 13).

1. Set up the first PCR reaction as follows:a. 1O!JL of lOX PCR bufferb. 1O!JL of lOX dNTP solutionc. 0.5 each of overlapping oligonucleotidesd. 2.5 U of Taq DNA polymerase;e. Sterile water to a final volume of 100 !JLf. Overlay sample with 50 !JL of sterile mineral oil

2. Amplify by PCR using the following cycle profile Notes 3, and 5):a. Initial denaturation 94C, 5 minb. 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 bufferb. 10!JL of lOX dNTP solutionc. 1!JL of first PCR reaction as templated. 1 of each flanking primere. 2.5 U Taq polymerasef. Sterile water to final volume of 100 !JLg. Overlay sample with 50 !JL of sterile mineral oil4. Run the second PCR using the following cycle profile:a. Initial denaturation 94C, 5 minb. 25 main cycles 55C, 1 minnoc, 1 min

94C, 1 minc. Final extension noc, 5 min5. Analyze 10 !JL of the first and second PCR reactions by agarose gel electrophoresis

Chapter 13). A faint smear should be present in the first PCR reaction, and a bandcorresponding to the size of the desired product should be present in the second PCRreaction Note 6).

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6126. Digest the product from the second PCR react ion and clone into a suitable vector

(see Note 7).

1. is not necessary to purify oligonucleotides when using this method. In addition, there isno 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 strandsand will extend inward toward each other.

3. The number of cycles needed for the first PCR reaction can be varied depending on thenumber 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 additionresults in the generation of many different-sized products that do not amplify well in thesecond PCR reaction.

5. It should be noted that the nucleic acid sequences of the over laps may inf luence theannealing 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 methodshould approximate that observed for other PCR protocols using Taq polymerase.

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., andAmheim, 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 syntheticgenes using the polymerase chain reaction. BioTechniques 9, 298-299.

4. Mullis, K., Faloona, F., Scharf, S., Saiki, R., Hom, G., and Erlich, H. (1986) Specificenzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold SpringHarbor Symp. Quant. BioI. 51, 263-273.

5. Dillon, P. J., Nelbock, P., Perkins, A., and Rosen, C. A. (1990) Function of the humanimmunodef iciency virus types I and 2 Rev proteins is dependent upon their ability tointeract 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. Acquired Immun. Defic. Syndrome 4, 558-567.