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OmniChange: the sequence independent method for simultaneous site-saturation of five codons.

Dennig A, Shivange AV, Marienhagen J, Schwaneberg U - PLoS ONE (2011)

Bottom Line: As proof of principle, five chemically cleaved DNA fragments, each carrying one NNK-degenerated codon, were generated and assembled to full gene length in a one-pot-reaction without additional PCR-amplification or use of restriction enzymes or ligases.Sequencing revealed the presence of up to 27 different codons at individual positions, corresponding to 84.4% of the theoretical diversity offered by NNK-degeneration.OmniChange is absolutely sequence independent, does not require a minimal distance between mutated codons and can be accomplished within a day.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.

ABSTRACT
Focused mutant library generation methods have been developed to improve mainly "localizable" enzyme properties such as activity and selectivity. Current multi-site saturation methods are restricted by the gene sequence, require subsequent PCR steps and/or additional enzymatic modifications. Here we report, a multiple site saturation mutagenesis method, OmniChange, which simultaneously and efficiently saturates five independent codons. As proof of principle, five chemically cleaved DNA fragments, each carrying one NNK-degenerated codon, were generated and assembled to full gene length in a one-pot-reaction without additional PCR-amplification or use of restriction enzymes or ligases. Sequencing revealed the presence of up to 27 different codons at individual positions, corresponding to 84.4% of the theoretical diversity offered by NNK-degeneration. OmniChange is absolutely sequence independent, does not require a minimal distance between mutated codons and can be accomplished within a day.

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Three steps in the primer design for the OmniChange method on the examples of positions E31 and T77.(A) Selection of targeted codons for NNK saturation (dark-grey highlighted letters). (B) Twelve nucleotides downstream of a targeted codon are selected as phosphorothiolated nucleotides for subsequent overhang generation by chemical cleavage into multiple small fragments. Remaining overhangs enable efficient hybridization of generated DNA fragments (light-grey highlighted letters). (C) Design of each oligonucleotide 3′end for successful PCR amplification of DNA fragments. Arrows indicate Phusion DNA Polymerase amplifcation direction. Italic letters are the phosphorothiolated nucleotides on the 5′ends of every primer used in the OmniChange method.
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pone-0026222-g003: Three steps in the primer design for the OmniChange method on the examples of positions E31 and T77.(A) Selection of targeted codons for NNK saturation (dark-grey highlighted letters). (B) Twelve nucleotides downstream of a targeted codon are selected as phosphorothiolated nucleotides for subsequent overhang generation by chemical cleavage into multiple small fragments. Remaining overhangs enable efficient hybridization of generated DNA fragments (light-grey highlighted letters). (C) Design of each oligonucleotide 3′end for successful PCR amplification of DNA fragments. Arrows indicate Phusion DNA Polymerase amplifcation direction. Italic letters are the phosphorothiolated nucleotides on the 5′ends of every primer used in the OmniChange method.

Mentions: Ten oligonucleotides were designed as PCR-primers for simultaneously saturating five codons (Table 2). Generally, oligonucleotides for OmniChange PCR should be designed to maximize product yields in PCRs (e.g. minimum 40% GC content) [17]. Reverse primers harboring a NNK codon were designed to have a GC content of minimum 40% and a Tm of preferably 63°C±3°C (calculated using OligoAnalyser tool at http://eu.idtdna.com). In addition, all oligonucleotides have at least 12 or 13 phosphorothioate nucleotide bonds at the 5′-terminus to generate single stranded overhangs for DNA hybridization after iodine cleavage [27] (Table 2). A detailed guidance for oligonucleotides design is described exemplary for positions E31 and T77 in Figure 3.


OmniChange: the sequence independent method for simultaneous site-saturation of five codons.

Dennig A, Shivange AV, Marienhagen J, Schwaneberg U - PLoS ONE (2011)

Three steps in the primer design for the OmniChange method on the examples of positions E31 and T77.(A) Selection of targeted codons for NNK saturation (dark-grey highlighted letters). (B) Twelve nucleotides downstream of a targeted codon are selected as phosphorothiolated nucleotides for subsequent overhang generation by chemical cleavage into multiple small fragments. Remaining overhangs enable efficient hybridization of generated DNA fragments (light-grey highlighted letters). (C) Design of each oligonucleotide 3′end for successful PCR amplification of DNA fragments. Arrows indicate Phusion DNA Polymerase amplifcation direction. Italic letters are the phosphorothiolated nucleotides on the 5′ends of every primer used in the OmniChange method.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198389&req=5

pone-0026222-g003: Three steps in the primer design for the OmniChange method on the examples of positions E31 and T77.(A) Selection of targeted codons for NNK saturation (dark-grey highlighted letters). (B) Twelve nucleotides downstream of a targeted codon are selected as phosphorothiolated nucleotides for subsequent overhang generation by chemical cleavage into multiple small fragments. Remaining overhangs enable efficient hybridization of generated DNA fragments (light-grey highlighted letters). (C) Design of each oligonucleotide 3′end for successful PCR amplification of DNA fragments. Arrows indicate Phusion DNA Polymerase amplifcation direction. Italic letters are the phosphorothiolated nucleotides on the 5′ends of every primer used in the OmniChange method.
Mentions: Ten oligonucleotides were designed as PCR-primers for simultaneously saturating five codons (Table 2). Generally, oligonucleotides for OmniChange PCR should be designed to maximize product yields in PCRs (e.g. minimum 40% GC content) [17]. Reverse primers harboring a NNK codon were designed to have a GC content of minimum 40% and a Tm of preferably 63°C±3°C (calculated using OligoAnalyser tool at http://eu.idtdna.com). In addition, all oligonucleotides have at least 12 or 13 phosphorothioate nucleotide bonds at the 5′-terminus to generate single stranded overhangs for DNA hybridization after iodine cleavage [27] (Table 2). A detailed guidance for oligonucleotides design is described exemplary for positions E31 and T77 in Figure 3.

Bottom Line: As proof of principle, five chemically cleaved DNA fragments, each carrying one NNK-degenerated codon, were generated and assembled to full gene length in a one-pot-reaction without additional PCR-amplification or use of restriction enzymes or ligases.Sequencing revealed the presence of up to 27 different codons at individual positions, corresponding to 84.4% of the theoretical diversity offered by NNK-degeneration.OmniChange is absolutely sequence independent, does not require a minimal distance between mutated codons and can be accomplished within a day.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.

ABSTRACT
Focused mutant library generation methods have been developed to improve mainly "localizable" enzyme properties such as activity and selectivity. Current multi-site saturation methods are restricted by the gene sequence, require subsequent PCR steps and/or additional enzymatic modifications. Here we report, a multiple site saturation mutagenesis method, OmniChange, which simultaneously and efficiently saturates five independent codons. As proof of principle, five chemically cleaved DNA fragments, each carrying one NNK-degenerated codon, were generated and assembled to full gene length in a one-pot-reaction without additional PCR-amplification or use of restriction enzymes or ligases. Sequencing revealed the presence of up to 27 different codons at individual positions, corresponding to 84.4% of the theoretical diversity offered by NNK-degeneration. OmniChange is absolutely sequence independent, does not require a minimal distance between mutated codons and can be accomplished within a day.

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