Tejeda Y, Fernandez JR, Colarte AB, Taylor CY

Language: Spanish
References: 10
Page: 164-167
PDF size: 301.04 Kb.

ABSTRACT

A common rate-limiting step in many high throughput proteome scale analyses is the cloning of predicted open reading frames (ORFs) into technique-specific vectors. For example, methodologies for the detection of protein interactions such as Yeast-two-hybrid (Y2H) assay require validation using alternative methods as Protein Fragment Complementation Assays (PCA) or pulldown experiments. Various experimental alternatives for rapid homologous recombination gene transfer in vivo between the Y2H and PCA vectors were evaluated. Two sets of universal primers sharing an overlap of 31 b homology between inserts and acceptor vectors were designed and PCR performance conditions were tested. Cotransformation of PCR products with the digested acceptor vector in E. coli strain allowed homologous recombination cloning. The method was proved to be effective for cloning 5 ORFs with sizes ranging from 0.294 to 1.2 kb. The proposed method allows the quick transfer of any open reading frames between the Y2H and PCA assay vector systems by using a universal set of primers. It doesn’t depend on the presence of specific restriction sites in the acceptor vector or causes changes in the open reading frame. This system will be useful for routine validation of protein interactions. We also report here the feasibility of DH10B strain for homologous recombination cloning.

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