Limits...
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.

Show MeSH
Obtained diversity by OmniChange shown as partial sequence alignment of 48 randomly “picked” clones.Partial sequence alignment of wild-type gene (template DNA) to 48 random clones from a multi site-saturation library generated by OmniChange. The five sites targeted for saturation are highlighted in white. Arrows indicate the distance between the targeted positions in base pairs. A comparison of the codons in the white columns shows the obtained diversity by saturating five independent positions simultaneously by OmniChange.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198389&req=5

pone-0026222-g002: Obtained diversity by OmniChange shown as partial sequence alignment of 48 randomly “picked” clones.Partial sequence alignment of wild-type gene (template DNA) to 48 random clones from a multi site-saturation library generated by OmniChange. The five sites targeted for saturation are highlighted in white. Arrows indicate the distance between the targeted positions in base pairs. A comparison of the codons in the white columns shows the obtained diversity by saturating five independent positions simultaneously by OmniChange.

Mentions: For detailed statistical analysis, 48 randomly picked clones were sent for forward and reverse sequencing. Multiple sequence alignments with the wild-type sequence confirmed that all five codons were fully saturated and no clone retained the original sequence (Figure 2 and Table 3). Of the 48 sequences analyzed, one wild-type codon could be found at two amino acid positions (E31 and G187) and two wild-type codons were found at position V298. At amino acid position T77 not a single wild-type codon was identified due to the fact that the NNK does not offer the respective wild-type codon [25]. Surprisingly, on position K139 one wild-type codon was identified (clone 8) after sequencing although NNK does not offer AAA as randomly introduced codon (Figure 2). In this case, we have to assume that either oligonucleotide synthesis was not performed completely error-free or exonuclease activity of Phusion polymerase corrected the mismatches during PCR. NNK degeneracy offers a diversity of 32 different codons at each position covering all canonical amino acids and avoids two of the three stop codons [26]. Twenty-one different codons at position E31 could be observed, representing already 65.6% of the possible diversity after sequencing of only 48 clones. For all other positions, even a higher coverage could be obtained (Table 3): T77 (26 unique codons 81.3%), K139 (24 unique codons 75.0%), G187 (27 unique codons 84.4%), V298 (27 unique codons 84.4% diversity). Site-specific cleavage of phosphorothioated nucleotides from 5′-ends of double-stranded DNA was developed as cloning technology reporting that the cleavage reaction did not cause any additional mutations [27]. Sequence analysis of the 48 clones also showed that the cleavage reaction (Step 2) and fragment hybridization (Step 3) did not introduce additional point mutations nor caused insertions or deletions. A mutation frequency of 0.17 per kb (11 mutations in 62.3 kb) was found within the 1.3 kb phytase gene at least 21 bp away from targeted positions which can likely be attributed to the employed polymerase.


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

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

Obtained diversity by OmniChange shown as partial sequence alignment of 48 randomly “picked” clones.Partial sequence alignment of wild-type gene (template DNA) to 48 random clones from a multi site-saturation library generated by OmniChange. The five sites targeted for saturation are highlighted in white. Arrows indicate the distance between the targeted positions in base pairs. A comparison of the codons in the white columns shows the obtained diversity by saturating five independent positions simultaneously by OmniChange.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026222-g002: Obtained diversity by OmniChange shown as partial sequence alignment of 48 randomly “picked” clones.Partial sequence alignment of wild-type gene (template DNA) to 48 random clones from a multi site-saturation library generated by OmniChange. The five sites targeted for saturation are highlighted in white. Arrows indicate the distance between the targeted positions in base pairs. A comparison of the codons in the white columns shows the obtained diversity by saturating five independent positions simultaneously by OmniChange.
Mentions: For detailed statistical analysis, 48 randomly picked clones were sent for forward and reverse sequencing. Multiple sequence alignments with the wild-type sequence confirmed that all five codons were fully saturated and no clone retained the original sequence (Figure 2 and Table 3). Of the 48 sequences analyzed, one wild-type codon could be found at two amino acid positions (E31 and G187) and two wild-type codons were found at position V298. At amino acid position T77 not a single wild-type codon was identified due to the fact that the NNK does not offer the respective wild-type codon [25]. Surprisingly, on position K139 one wild-type codon was identified (clone 8) after sequencing although NNK does not offer AAA as randomly introduced codon (Figure 2). In this case, we have to assume that either oligonucleotide synthesis was not performed completely error-free or exonuclease activity of Phusion polymerase corrected the mismatches during PCR. NNK degeneracy offers a diversity of 32 different codons at each position covering all canonical amino acids and avoids two of the three stop codons [26]. Twenty-one different codons at position E31 could be observed, representing already 65.6% of the possible diversity after sequencing of only 48 clones. For all other positions, even a higher coverage could be obtained (Table 3): T77 (26 unique codons 81.3%), K139 (24 unique codons 75.0%), G187 (27 unique codons 84.4%), V298 (27 unique codons 84.4% diversity). Site-specific cleavage of phosphorothioated nucleotides from 5′-ends of double-stranded DNA was developed as cloning technology reporting that the cleavage reaction did not cause any additional mutations [27]. Sequence analysis of the 48 clones also showed that the cleavage reaction (Step 2) and fragment hybridization (Step 3) did not introduce additional point mutations nor caused insertions or deletions. A mutation frequency of 0.17 per kb (11 mutations in 62.3 kb) was found within the 1.3 kb phytase gene at least 21 bp away from targeted positions which can likely be attributed to the employed polymerase.

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