Recursive PCR: a novel technique for total gene synthesis.

Key words: gene synthesis / human lysozyme / PCR / proteinengineering/recursive PCRIntroductionIn protein engineering applications involving mutagenesis andexpression of proteins from recombinant DNA, synthetic genesoffer many advantages over using cloned naturally occurringgenes. By precisely specifying the nucleotide sequence, optimalcodon usage for the expression host can be ensured and con-venient restriction sites incorporated as required, facilitatingcassette mutagenesis and subcloning. The synthesis of even arelatively small gene has generally been considered a difficultand time consuming task, best left to specialist laboratories. Inthis paper, a new cost and labour saving PCR technique isdescribed which greatly simplifies the process of gene synthesisand has the potential for the synthesis of significantly larger genesthan currently established techniques.Synthetic genes are conventionally assembled by concatena-tion of shorter oligonucleotides. Generally, both DNA strandsare completely synthesized as short overlapping oligonucleotideswhich are phosphorylated, annealed and ligated to generate thefull-length product (Edge et al., 1981; Ferretti et al., 1986; Belletal., 1988). The cost of the synthesis can be reduced bysynthesizing oligonucleotides representing the partial sequenceof each strand, and the gaps in the annealed product 'filled in'using DNA polymerase prior to ligation (Rink et al., 1984). Inpractice, both methods give a low yield of the full-length productand require amplification by cloning before any further manipula-tion of the synthesized gene.Recently, a PCR procedure has been described in which a 234base oligonucleotide was chemically synthesized and primers usedto amplify any full-length molecules that resulted from thechemical synthesis (Barnet and Erfle, 1990). While this procedureeffectively increases the length of sequence that can be synthesizeddirectly at a useful yield, the length is still relatively short incomparison with even a moderately sized structural gene. Genescan also be assembled by the method of 'splicing by overlapextension' (Higuchi etal., 1988), in which PCR products arepurified away from their amplifying primers and extended againsteach other to produce a larger product. This product is simul-taneously amplified by the inclusion of smaller flanking primers.We have developed a PCR technique for gene synthesis whichrequires neither phosphorylation nor ligation, gives high yieldsand has the potential for the total synthesis of much larger genesthan other established techniques. The cost of this method isrelatively low because only oligonucleotides representing thepartial sequence of each strand are chemically synthesized, asin the 'DNA polymerase filling-in' method. The oligonucleotidesare mixed and subjected to PCR such that those overlapping attheir 3' ends are extended to give longer double-strandedproducts. This is repeated for the double-stranded products untilthe full-sized gene is obtained (Figure 1). The full-length productis subsequently amplified by the outermost 5' oligonucleotidesof each strand, which are present at a higher concentration thanthe internal oligonucleotides and act as primers for the amplifica-tion. The complete synthesis of the gene results from the mutualextension of two halves which themselves result from the mutualextension of two halves and so on. This is functionally identicalto the technique of recursion in computer programming and wetherefore describe this method as 'recursive PCR'.