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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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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).
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pone-0064870-g003: 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).

Mentions: In Figure 3A we show a schematic representation of the two human ‘Required for Meiotic Nuclear Division 5 Homolog’ isoforms RMND5a (hRMD5a) and RMND5b (hRMD5b) and their protein domains. The similarity of both isoforms is not only reflected in their domain distribution but also on the amino acid residue level with 70% identical residues. We have fused both proteins to the C-terminus of GFP and examined their localization after transfection of HEK293 cells (Fig. 3C). Despite strong identity, both isoforms localize very differently in cells. RMND5a is distributed in the cytosol and in the nucleus of the cell (upper panel, left), whereas RMND5b is mostly present in the cytosol in vesicular structures (upper panel, right). To identify sequence elements responsible for the altered localization, we made several hybrid RMND5 fusion proteins using a SOMA-like method (Fig. 3B). The produced fusion proteins are schematically illustrated in Fig. 3C (upper panel) with RMND5a (hRMD5a; blue bar), RMND5b (hRMD5b; grey bar) and several hybrid proteins thereof. The length of RMND5b fragments in the hybrid proteins is depicted in numbers of amino acids (e.g. hRMD5b 1-140). The SOMA-based method is outlined in Figure 3B. Briefly, several N-terminal RMND5b fragments were PCR-amplified. Primer pairs were chosen to contain 25 bp of homologous region to the RMND5a replacement site (TP231fwd; TP233rev; TP234rev; see Table 2). The resulting PCR products were phosphorylated and used as megaprimers for mutagenesis with a plasmid encoding RMND5a as a template. This specific application exhibited a success rate between 10% and 40%. A similar method dubbed “overlap extension PCR” has been described recently [19]. We find that our modified SOMA-based protocol can be applied for cloning purposes in a similar fashion with high success rates.


SOMA: a single oligonucleotide mutagenesis and cloning approach.

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

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672168&req=5

pone-0064870-g003: 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).
Mentions: In Figure 3A we show a schematic representation of the two human ‘Required for Meiotic Nuclear Division 5 Homolog’ isoforms RMND5a (hRMD5a) and RMND5b (hRMD5b) and their protein domains. The similarity of both isoforms is not only reflected in their domain distribution but also on the amino acid residue level with 70% identical residues. We have fused both proteins to the C-terminus of GFP and examined their localization after transfection of HEK293 cells (Fig. 3C). Despite strong identity, both isoforms localize very differently in cells. RMND5a is distributed in the cytosol and in the nucleus of the cell (upper panel, left), whereas RMND5b is mostly present in the cytosol in vesicular structures (upper panel, right). To identify sequence elements responsible for the altered localization, we made several hybrid RMND5 fusion proteins using a SOMA-like method (Fig. 3B). The produced fusion proteins are schematically illustrated in Fig. 3C (upper panel) with RMND5a (hRMD5a; blue bar), RMND5b (hRMD5b; grey bar) and several hybrid proteins thereof. The length of RMND5b fragments in the hybrid proteins is depicted in numbers of amino acids (e.g. hRMD5b 1-140). The SOMA-based method is outlined in Figure 3B. Briefly, several N-terminal RMND5b fragments were PCR-amplified. Primer pairs were chosen to contain 25 bp of homologous region to the RMND5a replacement site (TP231fwd; TP233rev; TP234rev; see Table 2). The resulting PCR products were phosphorylated and used as megaprimers for mutagenesis with a plasmid encoding RMND5a as a template. This specific application exhibited a success rate between 10% and 40%. A similar method dubbed “overlap extension PCR” has been described recently [19]. We find that our modified SOMA-based protocol can be applied for cloning purposes in a similar fashion with high success rates.

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