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SOMA: a single oligonucleotide mutagenesis and cloning approach.

Pfirrmann T, Lokapally A, Andréasson C, Ljungdahl P, Hollemann T - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Institute for Physiological Chemistry, Martin-Luther University Halle-Wittenberg, Halle, Germany. thorsten.pfirrmann@medizin.uni-halle.de

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.

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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).
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pone-0064870-g002: 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).

Mentions: SOMA is a technique for the site-directed mutagenesis of plasmids including substitutions, deletions and insertions. Additionally, the insertion feature can be employed to clone and shuffle DNA fragments. We routinely use SOMA to introduce mutations at single and multiple positions with success rates up to 90% depending on the primer design as assessed by diagnostic restriction digestion analysis of individual clones after primary transformation. The basic methodology is outlined in Fig. 1 and a specific application is schematically depicted in Fig. 2B. Briefly, a mutagenic primer complementary to the target sequence is designed to carry the desired mutation. It can either be directly synthesized with a 5′ phosphate group or it can be phosphorylated as described in the Methods section. In a thermocycler the mutagenic primer is annealed to the plasmid template, extended with Phusion High-Fidelity DNA polymerase and the fully extended product is made circular by ligation using Taq DNA ligase. Following 30 cycles of amplification, the template is removed by DpnI digestion and the circular, single stranded mutagenized plasmid is used for E.coli transformation. After appropriate selection the plasmids are isolated and subjected to diagnostic restriction digestion or DNA sequencing. Standard thermocycler conditions are presented in Fig. 1. SOMA is based on Phusion High-Fidelity DNA polymerase that is a proofreading polymerase with extremely high extension rates, thus making the method suitable also for very large plasmids. To this end we have successfully mutagenized pBR322-derived plasmids as large as 14.3 kb. Out of 4 clones analyzed, 1 contained the desired substitution mutation as scored by diagnostic restriction analysis facilitated by the introduction of an HaeII restriction site together with the substitution mutation. To demonstrate the versatility of SOMA we present several applications.


SOMA: a single oligonucleotide mutagenesis and cloning approach.

Pfirrmann T, Lokapally A, Andréasson C, Ljungdahl P, Hollemann T - PLoS ONE (2013)

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).
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pone-0064870-g002: 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).
Mentions: SOMA is a technique for the site-directed mutagenesis of plasmids including substitutions, deletions and insertions. Additionally, the insertion feature can be employed to clone and shuffle DNA fragments. We routinely use SOMA to introduce mutations at single and multiple positions with success rates up to 90% depending on the primer design as assessed by diagnostic restriction digestion analysis of individual clones after primary transformation. The basic methodology is outlined in Fig. 1 and a specific application is schematically depicted in Fig. 2B. Briefly, a mutagenic primer complementary to the target sequence is designed to carry the desired mutation. It can either be directly synthesized with a 5′ phosphate group or it can be phosphorylated as described in the Methods section. In a thermocycler the mutagenic primer is annealed to the plasmid template, extended with Phusion High-Fidelity DNA polymerase and the fully extended product is made circular by ligation using Taq DNA ligase. Following 30 cycles of amplification, the template is removed by DpnI digestion and the circular, single stranded mutagenized plasmid is used for E.coli transformation. After appropriate selection the plasmids are isolated and subjected to diagnostic restriction digestion or DNA sequencing. Standard thermocycler conditions are presented in Fig. 1. SOMA is based on Phusion High-Fidelity DNA polymerase that is a proofreading polymerase with extremely high extension rates, thus making the method suitable also for very large plasmids. To this end we have successfully mutagenized pBR322-derived plasmids as large as 14.3 kb. Out of 4 clones analyzed, 1 contained the desired substitution mutation as scored by diagnostic restriction analysis facilitated by the introduction of an HaeII restriction site together with the substitution mutation. To demonstrate the versatility of SOMA we present several applications.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Institute for Physiological Chemistry, Martin-Luther University Halle-Wittenberg, Halle, Germany. thorsten.pfirrmann@medizin.uni-halle.de

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.

Show MeSH
Related in: MedlinePlus