Pfirrmann T et al. (2013), SOMA: a single oligonucleotide mutagenesis and ...

XB-ART-48120

PLoS One 2013 Jun 04;86:e64870. doi: 10.1371/journal.pone.0064870.

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SOMA: a single oligonucleotide mutagenesis and cloning approach.

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

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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.

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

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	Figure 2. Standard primer for SOMA-construction of a GFP reporter (A).Phosphorylated primer (red P) with diagnostic EcoRI site (red C; EcoRI*), 23 bp of GFP (green sequence) and morpholino target sequence in frame with GFP (NNN)x including ATG (red sequence). Green fluorescent protein (GFP) gene (green); ampicillin resistance gene (AmpR; yellow); URA3 gene, yeast centromere (CEN), autonomous replication sequence (ARS) (red). Schematic outline of the SOMA method for production of GFP reporters (B). (1) denature pTP218; (2) 5′ phosphorylated single primer anneals; (3) primer is extended to the 5′ end; (4) nicks are ligated; (5) template strand is digested with DpnI; (6) mutated single stranded plasmid transformed into E. coli and positive clones identified by lack of EcoRI site. Test of TRIM2 GFP reporter construct in vivo (C). Synthetic reporter sense RNA injected into fertilized Xenopus laevis embryos at the one-cell stage; TRIM2- or Standard (STRD)-morpholino injected into one blastomere at the two-cell stage. Visualization after 1 day by brightfield or fluorescence microscopy (GFP). 99 of total 101 standard morpholino (STRD) injected embryos showed a GFP signal on both sides (98%); 249 of total 271 Trim2- morpholino injected embryos showed a GFP signal on one side (92%) (right).
	Figure 3. Restriction site independent cloning.(A) Schematic representation of two human RMND5 isoforms (hRMD5a, hRMD5b) with domains; numbers indicate amino acids. (B) Schematic outline of RMND5a/b chimera production. (1) pTP224 as template; (2) PCR with primer P1 and P2 resulting in RMND5b fragment (3) Phosphorylation of 1st PCR product. (4) 5′ phosphorylated PCR product anneals; (5) is extended to the 5′ end; (6) nicks are ligated; (7) template strand is digested with DpnI; (8) product transformed into E. coli. Green fluorescent protein (GFP) gene (green); Kanamycine resistance gene (Kan/Neo; orange); RMND5b (grey); RMND5a (blue) (C) Localization of hRMD5a (blue), hRMD5b (grey) and different hybrid fusion (blue/grey) in HEK293 cells. Length of RMD5b fragments indicated as numbers of amino acids (e.g. hRMD5b 1-140).
	Figure 4. Generation of a semi-random mutagenized gene library using SOMA.(A) Ssy5 sequence with autolytic processing site (scissors) between A381 and A382 (blue). Replacement of conserved isoleucine378 (I; green) with aspartate (D; red) inactivates Ssy5. Primer D378X with random codon (NNN) theoretically capable of encoding all amino acids at position 378 (green). (B) Table with active D378 substitutions recovered in plasmids selected based on their ability to confer Ssy5-dependent growth on YPD+MM. (C) Dilutions of strain HKY77 (ssy5Δ) carrying pSH120 (SSY5; +), pRS316 (empty plasmid; -), I378G, I378D, I378S, I378F, I378V, or I378L.
	Figure 1. Flow scheme of the SOMA method (left).A mutagenesis primer is phosphorylated 5′ and used for a PCR reaction. Phusion polymerase amplifies the mutant strand, Taq Ligase ligates the nicks during the reaction. A DpnI digest leaves the mutagenized single stranded plasmid that is directly transformed into E. coli for selection and plasmid isolation. PCR condition for SOMA (right).

References [+] :

Andréasson, Receptor-mediated endoproteolytic activation of two transcription factors in yeast. 2002, Pubmed