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XB-ART-48120
PLoS One January 1, 2014; 8 (6): e64870.
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SOMA: a single oligonucleotide mutagenesis and cloning approach.

Pfirrmann T , Lokapally A , Andréasson C , Ljungdahl P , Hollemann T .


Abstract
Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA) that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.

PubMed ID: 23750217
PMC ID: PMC3672168
Article link: PLoS One


Species referenced: Xenopus laevis
Genes referenced: rmnd5a tff3.7 trim2


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References [+] :
Andréasson, Receptor-mediated endoproteolytic activation of two transcription factors in yeast. 2002, Pubmed