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.
Müller
Kristian M
KM
Institut für Biologie III, Universität Freiburg Schänzlestrasse 1, 79104 Freiburg, Germany. kristian@biologie.uni-freiburg.de
Stebel
Sabine C
SC
Knall
Susanne
S
Zipf
Gregor
G
Bernauer
Hubert S
HS
Arndt
Katja M
KM
D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D053938:DNA Fragmentation; D032621:DNA Shuffling / Q000379:methods*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D009711:Nucleotides / Q000378:metabolism; D010880:Piperidines / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012984:Software; D019914:Taq Polymerase / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism*
D003856:Deoxyuracil Nucleotides; D009711:Nucleotides; D010880:Piperidines; D013942:Thymine Nucleotides; C027078:deoxyuridine triphosphate; C032727:piperidine; D015500:Chloramphenicol O-Acetyltransferase; D019914:Taq Polymerase; C070331:Tli polymerase; D004259:DNA-Directed DNA Polymerase; D043224:Deoxyribonuclease (Pyrimidine Dimer); C024157:thymidine 5'-triphosphate