Creation of libraries with long ORFs by polymerization of a microgene

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Abstract
We describe a novel method for constructing pools of DNA sequences that encode large proteins with molecular diversity. Sets of primer pairs that form 8 to 10 complementary base pairs in the 3′ region and have double mismatch pairs at their 3′-OH ends were designed so that primer dimers recreated short stretches of DNA (microgenes) devoid of termination codons. Cycles of denaturation and elongation reactions with a pair of primers, four dNTPs, and 3′–5′ exo+ thermostable DNA polymerase gave head-to-tail polymers of the primer dimer unit (microgene) whose sizes exceeded 12 kb. No template was required in this reaction, but mismatched nucleotides at 3′-OH ends of the primers were critical for efficient polymerization. At end-joining junctions of a microgene, nucleotide insertions and deletions randomly occurred, resulting in combinatorial libraries of three reading frames from a single microgene. Further molecular diversity could be incorporated by using a mixture of primers. The resultant polymers have long ORFs whose products have a repetitious nature that could facilitate the formation of higher structures of translated products. Thus, microgene polymers may be used as a source of libraries for in vitro protein evolution experiments. Ligation of a microgene is apparently related to the nonhomologous recombination of double-strand breaks in DNA that has been shown to be catalyzed by DNA polymerases. We named this polymerization reaction the “microgene polymerization reaction.”