Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone sub-sequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their res-pective likelihood of occurrence, taking the guess-work out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyl-transferase gene libraries. A 33 % dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a frag-ment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs