Model-guided ligation strategy for optimal assembly of DNA libraries.
Protein Eng Des Sel
; 25(10): 669-78, 2012 Oct.
Article
em En
| MEDLINE
| ID: mdl-22543356
ABSTRACT
DNA ligation is essential to many molecular biology manipulations, but this reaction is often carried out by following generic guidelines or by trial and error. Maximizing the desired ligation product is especially important in DNA library construction for directed evolution experiments since library diversity is directly affected by ligation efficiency. Here, we suggest that display vectors that rely on Type IIP restriction sites for cloning should be redesigned to utilize Type IIS restriction sites instead because ligation yield is significantly improved we observed up to 15- and 2.6-fold increases in desired products for circular and linear ligation reactions, respectively. To guide ligation optimization more rationally, we developed an easily parameterized thermodynamic model that predicts product distributions based on input DNA concentrations and free energies of the ligation events. We applied this model to study ligation reactions using a ribosome display vector redesigned with Type IIS restriction sites (pRDV2). We computationally predicted and experimentally validated the relative abundance of various products in three-piece linear ligations as well as the extent of transformation from vector-insert circular ligations. Based on our results, we provide general insights into ligation and we outline guidelines for optimizing this reaction for both in vivo and in vitro display methodologies.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Biblioteca Gênica
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Protein Eng Des Sel
Assunto da revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Estados Unidos