title: Directed PCR-free engineering of highly repetitive DNA sequences.
abstract: BACKGROUND
  Highly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A)) tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products.
  
  
  RESULTS
  For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays.
  
  
  CONCLUSIONS
  Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.
authors.affiliation: Molecular Microbiology, Department of Biology, University of Konstanz, 78457 Konstanz, Germany. elke.deuerling@uni-konstanz.de
authors.forename: Annika
authors.initials: A
authors.lastname: Scior
authors.forename: Steffen
authors.initials: S
authors.lastname: Preissler
authors.forename: Miriam
authors.initials: M
authors.lastname: Koch
authors.forename: Elke
authors.initials: E
authors.lastname: Deuerling
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005818:Genetic Engineering; D006639:Histidine; D015151:Immunoblotting; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D025842:SUMO-1 Protein; D012995:Solubility
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C097188:polyglutamine; D006639:Histidine; D004247:DNA