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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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The 4-step strategy for the simultaneous saturation of 5 independent codons by OmniChange.OmniChange comprises four steps: Step 1. Amplification of five DNA fragments bearing a NNK-saturated codon (indicated with *). Step 2. Chemical cleavage to generate complementary single-stranded 5′-overhangs. Step 3. Hybridization of all fragments to a full circular plasmid containing ten DNA nicks. Step 4. Transformation and nick-repair in E. coli BL21-Gold (DE3) lacIQ1.
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pone-0026222-g001: The 4-step strategy for the simultaneous saturation of 5 independent codons by OmniChange.OmniChange comprises four steps: Step 1. Amplification of five DNA fragments bearing a NNK-saturated codon (indicated with *). Step 2. Chemical cleavage to generate complementary single-stranded 5′-overhangs. Step 3. Hybridization of all fragments to a full circular plasmid containing ten DNA nicks. Step 4. Transformation and nick-repair in E. coli BL21-Gold (DE3) lacIQ1.

Mentions: The OmniChange multi site-saturation method consists of four steps: Step 1. Fragment generation by PCR with oligonucleotides containing NNK-codons, Step 2. DNA-cleavage reaction for the generation of complementary 5′-overhangs, Step 3. Assembly via DNA-hybridization and Step 4. Transformation and nick repair by E. coli BL21-Gold (DE3) lacIQ1 (Figure 1).


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

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

The 4-step strategy for the simultaneous saturation of 5 independent codons by OmniChange.OmniChange comprises four steps: Step 1. Amplification of five DNA fragments bearing a NNK-saturated codon (indicated with *). Step 2. Chemical cleavage to generate complementary single-stranded 5′-overhangs. Step 3. Hybridization of all fragments to a full circular plasmid containing ten DNA nicks. Step 4. Transformation and nick-repair in E. coli BL21-Gold (DE3) lacIQ1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026222-g001: The 4-step strategy for the simultaneous saturation of 5 independent codons by OmniChange.OmniChange comprises four steps: Step 1. Amplification of five DNA fragments bearing a NNK-saturated codon (indicated with *). Step 2. Chemical cleavage to generate complementary single-stranded 5′-overhangs. Step 3. Hybridization of all fragments to a full circular plasmid containing ten DNA nicks. Step 4. Transformation and nick-repair in E. coli BL21-Gold (DE3) lacIQ1.
Mentions: The OmniChange multi site-saturation method consists of four steps: Step 1. Fragment generation by PCR with oligonucleotides containing NNK-codons, Step 2. DNA-cleavage reaction for the generation of complementary 5′-overhangs, Step 3. Assembly via DNA-hybridization and Step 4. Transformation and nick repair by E. coli BL21-Gold (DE3) lacIQ1 (Figure 1).

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.

Show MeSH
Related in: MedlinePlus