title: Patch cloning method for multiple site-directed and saturation mutagenesis.
abstract: BACKGROUND
  Various DNA manipulation methods have been developed to prepare mutant genes for protein engineering. However, development of more efficient and convenient method is still demanded. Homologous DNA assembly methods, which do not depend on restriction enzymes, have been used as convenient tools for cloning and have been applied to site-directed mutagenesis recently. This study describes an optimized homologous DNA assembly method, termed as multiple patch cloning (MUPAC), for multiple site-directed and saturation mutagenesis.
  
  
  RESULTS
  To demonstrate MUPAC, we introduced five back mutations to a mutant green fluorescent protein (GFPuv) with five deleterious mutations at specific sites and transformed Escherichia coli (E. coli) with the plasmids obtained. We observed that the over 90% of resulting colonies possessed the plasmids containing the reverted GFPuv gene and exhibited fluorescence. We extended the test to introduce up to nine mutations in Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RT) by assembling 11 DNA fragments using MUPAC. Analysis of the cloned plasmid by electrophoresis and DNA sequencing revealed that approximately 30% of colonies had the objective mutant M-MLV RT gene. Furthermore, we also utilized this method to prepare a library of mutant GFPuv genes containing saturation mutations at five specific sites, and we found that MUPAC successfully introduced NNK codons at all five sites, whereas other site remained intact.
  
  
  CONCLUSIONS
  MUPAC could efficiently introduce various mutations at multiple specific sites within a gene. Furthermore, it could facilitate the preparation of experimental gene materials important to molecular and synthetic biology research.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Taniguchi
authors.forename: Sayumi
authors.initials: S
authors.lastname: Nakayama
authors.forename: Takashi
authors.initials: T
authors.lastname: Kawakami
authors.affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan. murah@bio.c.u-tokyo.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Murakami
mesh_terms: D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon; D004247:DNA / Q000235:genetics*; D053938:DNA Fragmentation; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009052:Leukemia Virus, Murine / Q000201:enzymology / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D003062:Codon; D049452:Green Fluorescent Proteins; D004247:DNA; D012194:RNA-Directed DNA Polymerase