Directed PCR-free engineering of highly repetitive DNA sequences

Scior, Annika; Preissler, Steffen; Koch, Miriam; Deuerling, Elke

Directed PCR-free engineering of highly repetitive DNA sequences

Dateien

deuerling_koch_etal.pdfGröße: 838.82 KBDownloads: 290

Datum

2011

Autor:innen

Scior, Annika

Preissler, Steffen

Koch, Miriam

Deuerling, Elke

Open Access-Veröffentlichung

Open Access Gold

Sammlungen

Biologie

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen in

BMC Biotechnology. 2011, 11(1), 87. eISSN 1472-6750. Available under: doi: 10.1186/1472-6750-11-87

Zusammenfassung

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.
Conclusion: 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.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

Zitieren

ISO 690

SCIOR, Annika, Steffen PREISSLER, Miriam KOCH, Elke DEUERLING, 2011. Directed PCR-free engineering of highly repetitive DNA sequences. In: BMC Biotechnology. 2011, 11(1), 87. eISSN 1472-6750. Available under: doi: 10.1186/1472-6750-11-87

BibTex

@article{Scior2011Direc-16812,
  year={2011},
  doi={10.1186/1472-6750-11-87},
  title={Directed PCR-free engineering of highly repetitive DNA sequences},
  number={1},
  volume={11},
  journal={BMC Biotechnology},
  author={Scior, Annika and Preissler, Steffen and Koch, Miriam and Deuerling, Elke},
  note={Article Number: 87}
}

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    <dcterms:abstract xml:lang="eng">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.&lt;br /&gt;Results: For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by&lt;br /&gt;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&lt;br /&gt;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.&lt;br /&gt;Conclusion: Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly&lt;br /&gt;repetitive DNA sequences of defined lengths by simple standard cloning procedures.</dcterms:abstract>
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Universitätsbibliographie

Ja