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

ABSTRACT: 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

SUBMITTER: Muller KM 

PROVIDER: S-EPMC1182171 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature