Versatile DNA fragmentation and directed evolution with nucleotide exchange and excision technology.

Mimicking natural evolution by DNA shuffling is a commonly used method for the optimization of DNA and protein properties. Here, we present an advancement of this approach whereby a gene library is amplified using a standard polymerase chain reaction (PCR), but incorporates dUTP as a fragmentation-defining exchange nucleotide, together with the four standard dNTPs. Incorporated uracil bases are excised using uracil-DNA-glycosylase, and the DNA backbone subsequently is cleaved with piperidine. This oligonucleotide pool is then reassembled with an internal primer extension procedure using a proofreading polymerase to increase yield, and, finally, is amplified by PCR. Denaturing polyacrylamide urea gels demonstrate this method to produce adjustable fragmentation size ranges dependent on the dUTP:dTTP ratios. Using the model protein, chloramphenicol acetyltransferase I, the sequencing of shuffled gene libraries based on a PCR containing 33% dUTP revealed a low mutation rate, of approx 0.1%, with an average parental fragments size of 86 bases, even without the use of a fragment-size separation. Nucleotide exchange and excision technology (NExT) DNA shuffling is, thus, reproducible and easily executed, making it superior to competing techniques. Additionally, NExT fragmentation outcome can be predicted using the computer software, NExTProg.

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

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 subsequently 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 respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment 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.

Nucleotide exchange and excision technology DNA shuffling and directed evolution.

Remarkable success in optimizing complex properties within DNA and proteins has been achieved by directed evolution. In contrast to various random mutagenesis methods and high-throughput selection methods, the number of available DNA shuffling procedures is limited, and protocols are often difficult to adjust. The strength of the nucleotide exchange and excision technology (NExT) DNA shuffling described here is the robust, efficient, and easily controllable DNA fragmentation step based on random incorporation of the so-called 'exchange nucleotides' by PCR. The exchange nucleotides are removed enzymatically, followed by chemical cleavage of the DNA backbone. The oligonucleotide pool is reassembled into full-length genes by internal primer extension, and the recombined gene library is amplified by standard PCR. The technique has been demonstrated by shuffling a defined gene library of chloramphenicol acetyltransferase variants using uridine as fragmentation defining exchange nucleotide. Substituting 33% of the dTTP with dUTP in the incorporation PCR resulted in shuffled clones with an average parental fragment size of 86 bases and revealed a mutation rate of only 0.1%. Additionally, a computer program (NExTProg) has been developed that predicts the fragment size distribution depending on the relative amount of the exchange nucleotide.