Chimeric gene library construction by a simple and highly versatile method using recombination-dependent exponential amplification.
A simple and efficient method for the construction of chimeric gene libraries termed RDA-PCR (recombination-dependent exponential amplification polymerase chain reaction) was developed by modifying polymerase chain reaction. A chimeric gene library is generated from homologous parental genes with additional primer-annealing sequences at their "heads" and "tails". Two primers ("skew primers") are designed to exclusively anneal to either the heads of maternal genes or the tails of paternal genes. During the RDA-PCR, short annealing/extension periods facilitate homologous recombination. The chimeric sequences can be exponentially amplified to form the chimeric gene library, whereas parental sequences without crossovers are not amplified. As a model, we constructed a chimeric gene library of yellow and green fluorescent protein (yfp and gfp, respectively). The crossover point profile of RDA-PCR clones was compared with those obtained by (modified) family shuffling. PCR restriction fragment polymorphism (PCR-RFLP) analysis of the RDA-PCR clones showed a high content of chimeric genes in the library, whereas family shuffling required the modification using skew primers for selective enrichment of chimeric sequences. PCR-RFLP analysis also indicated that the crossover points of RDA-PCR chimeras were distributed over the entire protein-coding region. Moreover, as few as 2 bp of the continual identity of nucleotides were found at the crossover points at high frequency (30% of the tested clones), suggesting that RDA-PCR resulted in a higher diversity in crossover points than family shuffling.
Ikeuchi
Akinori
A
Laboratory of Molecular Biotechnology, Division of Molecular and Cellular Mechanisms, Graduate School of Bio- and Agricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Kawarasaki
Yasuaki
Y
Shinbata
Tomoya
T
Yamane
Tsuneo
T
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D005240:Feasibility Studies; D005784:Gene Amplification / Q000235:genetics; D015723:Gene Library; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins