In the primers shown in Table 1, all of the complementary bases have been added to one of the two primers (primers ‘b,’ ‘e’ and ‘g’), rather than adding some sequence to each primer. This way, the other primers (‘c,’ ‘d’ and ‘f’) can potentially be used with new SOEing primers (analogous to ‘b,’ ‘d’ and ‘g’) to join these fragments to other genes. Since the two templates share three nucleotides in primers ‘f’ and ‘g,’ these nucleotides contribute both to the overlap and to the priming portion of oligomer ‘g’ (they are both underlined and asterisked in Table 1). The portion of oligomer ‘e’ in parentheses is not related to either template, and does not contribute to the overlap. This is an example of insertional mutageneis (6) being carried out simultaneously with recombination.
As an example of the SOEing process, the complementary regions ‘d’ and ‘e’ containing the sequences which lead to the PCR products AD and EH having overlapping ends are shown in Figure 2.
PCR and SOE reactions were carried out in a thermocycler (Perkin-Elmer Cetus, Norwalk, CT) for 25 cycles, each consisting of 1 min at 94°C, 2 min at 50°C, and 3 min at 72°C. (The reaction probably produces all of the product in fewer than 25 cycles, but we have not examined the minimum number of cycles required. Extra cycles do not appear to cause any problems.) Taq polymerase was from PerkinElmer Cetus, and the reaction buffer was as recommended by the supplier (50 mM KCl, 10 mM Tris-Cl, pH 8.3, 1.5 mM MgCl2, 0.01% (wt/vol) gelatin). Deoxyribonucleotides were used at a final concentration of 200 µM. The buffer and deoxynucleoside triphosphates were each made as a 10x stock, and 10 µl were used per 100 µl reaction. One-half µl of polymerase (2.5 U) was used per reaction. Reactions were covered with mineral oil before thermal cycling.Purification of Fragments
PCR and SOEn products which were to be used as templates in further reactions were purified by electrophoresis through agarose (1% SeaKem LE agarose + 2% NuSieve GTG agarose, FMC BioProducts, Rockland, ME) in TAE buffer (0.04 M Tris-acetate, 0.001 M EDTA) with 0.5 µg/ml ethidium bromide in the gel. DNA from the appropriate bands was recovered from the gel fragment by GeneClean (Bio 101, La Jolla, CA). The final recombinant product was similarly gel-purified before cloning.Cloning of Fragments
The SOEn products were cut with restriction enzymes Sall and Xhol and ligated into the corresponding position of a pUC-derived plasmid which has been designed to act as an expression vector for class I MHC antigen binding regions, as described elsewhere (13).Analysis of Products
The cloned product was sequenced from the double-stranded template using a Sequenase kit (United States Biochemical, Cleveland, OH) with a modified protocol (9).Results Production of Fragments
Figure 3 shows the strategy for construction of the hybrid class 1/class II gene. Part A shows the original genes and the locations of the primers, while part B shows the strategy for SOEing these PCR products together. Figure 4 is a photograph of an ethidium bromide stained gel of the products of these reactions. The PCR products AB, CD, EF and GH, as well as the intermediate SOEn products AD and EH, are the major products of the reactions, though some byproduct bands are visible. These other products were eliminated in the gel purification step. Also, some additional products are visible in the final product lane (AH), but the band of the expected size is the major product.
Analysis of Product
The sequence of the cloned product is shown in Figure 5. Of approximately 1700 bases sequenced (coding regions from two clones), there was one unplanned mutation, presumably caused by misincorporation by the polymerase. This is compatible with the error frequency of 0.06% reported earlier (7).
Quite complicated constructions are possible with this approach. For example, we have used it to create and express a gene for a fusion protein in which the a helixes are replaced by the corresponding segments of a similar gene (7). The construction used to illustrate the technique in the present report is similarly complicated. In this construction, four gene segments from three different genes were SOEn together, two at a time, to produce the chimeric product. Simultaneously, a 15-base pair (bp) segment encoding a portion of the class I gene which was in a different exon from the one that was amplified was added at the recombination joint. We have thus accomplished recombination and insertional mutagenesis in one step.