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A method for high-throughput production of sequence-verified DNA libraries and strain collections.
Smith, Justin D; Schlecht, Ulrich; Xu, Weihong; Suresh, Sundari; Horecka, Joe; Proctor, Michael J; Aiyar, Raeka S; Bennett, Richard A O; Chu, Angela; Li, Yong Fuga; Roy, Kevin; Davis, Ronald W; Steinmetz, Lars M; Hyman, Richard W; Levy, Sasha F; St Onge, Robert P.
Afiliação
  • Smith JD; Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
  • Schlecht U; Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
  • Xu W; Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
  • Suresh S; Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
  • Horecka J; Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
  • Proctor MJ; Department of Surgery, Harvard Medical School and Massachusetts General Hospital, Boston, MA, USA.
  • Aiyar RS; Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
  • Bennett RA; Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
  • Chu A; Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
  • Li YF; Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
  • Roy K; Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
  • Davis RW; Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
  • Steinmetz LM; Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA.
  • Hyman RW; Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
  • Levy SF; Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY, USA.
  • St Onge RP; Department of Biochemistry and Cellular Biology, Stony Brook University, Stony Brook, NY, USA.
Mol Syst Biol ; 13(2): 913, 2017 02 13.
Article em En | MEDLINE | ID: mdl-28193641
ABSTRACT
The low costs of array-synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost-effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site-specific recombination to index library DNA, and next-generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost-effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Leveduras / Análise de Sequência de DNA Idioma: En Revista: Mol Syst Biol Assunto da revista: BIOLOGIA MOLECULAR / BIOTECNOLOGIA Ano de publicação: 2017 Tipo de documento: Article País de afiliação: Estados Unidos
Texto completo
Imprimir
XML
PubMed Links
Buscar no Google

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Leveduras / Análise de Sequência de DNA Idioma: En Revista: Mol Syst Biol Assunto da revista: BIOLOGIA MOLECULAR / BIOTECNOLOGIA Ano de publicação: 2017 Tipo de documento: Article País de afiliação: Estados Unidos