Answers

1. Where can I find the IUPAC nucleotide ambiguity codes?

We recommend this site: http://www.chick.manchester.ac.uk/SiteSeer/IUPAC_codes.html

2. What should a degenerated library look like for the best chance of finding an improved version of my protein?

The answer depends on the specifics of your project. In general, it is advantageous to keep the diversity of a library as low as possible, targeting only the regions of a gene/protein that are likely to be functionally important. The following information can help determine this: crystal structure, conserved motifs, presence of homologs, etc. Feel free to contact us and benefit from our vast experience.

3. What are the advantages of synthetic libraries over conventional protocols for creating diversity?

Conventional protocols for degenerated library creation (e.g., error-prone PCR) incorporate many unwanted mutations. Moreover, methods like DNA shuffling cannot typically cause recombination of directly adjacent mutations. Synthetic combinatorial libraries, on the other hand, limit the introduction of mutations to defined regions at the precise frequencies requested. In addition, adjacent mutations will be recombined (shuffled) independent of their proximity.

4. Why do I need rational diversity when I can screen a large number of random mutants anyway?

There are a number of reasons:

  • Because you already know that certain amino acids substitutions disturb the function of your protein (e.g., cysteines in complementarity determining regions (CDRs).
  • Because the number of possible variants of a protein is astronomically high, exceeding the capacity of even the highest-throughput screening capabilities by many orders of magnitude. The fewer useless mutations, such as those occurring in less important regions of the protein or that cause frameshift or stop codons, the better your chances of finding a variant that results in the desired phenotype.
  • Some screening assays are cost and labor intensive; thus, screening fewer clones saves time and money.

5. What types of degenerated libraries are available?

  • Combinatorial Libraries (up to 1011 variants)
  • Simultaneous randomization of multiple codons
  • Site-Saturation Mutagenesis (up to 20 variants)
  • Randomization of a single codon with every possible non–wild type variant
  • Sequential Permutation Libraries (# of codons x 20 variants) 
  • Successive site-saturation mutagenesis
  • Controlled Randomization (up to 1011 variants)
  • Unbiased random substitutions with defined frequency
  • Cloned cDNA of all known human SH3 domains
  • Truncation Libraries
  • Customer defined truncations without out of frame mutations.

6. What material do I have to supply for degenerated library synthesis?

No material at all. All we need for library creation is the sequence file, submitted electronically, and information about the position and nature of the sites you want to randomize. We can provide a quote for your project through our online ordering system. If needed, you can then relate detailed information about your library request to our production scientists prior to starting the project.

7. Can a library be subcloned in my vector?

Yes. For this purpose customers either sent their own, individual vector or sent us a commercially available vector.

8. How are libraries supplied?

Two standard options are available:

  1. As a set of linear DNA fragments, ready for restriction enzyme digestion and subcloning.
  2. Subcloned into any vector and delivered as a glycerol stock and plasmid preparation.

9. What are some examples of what directed evolution can be used for?

  • Increase or adjust promoter strength or specificity
  • Enhance or modulate protein stability
  • Modify or combine enzyme properties
  • Increase binding affinities of receptors, ligands and antibodies
  • Optimize or alter signal peptide efficiencies
  • Destroy protein function while retaining immunogenicity
  • Combine and select natural polymorphisms
  • Increase protein half life
  • Adjust thermal stability

10. What quality criteria can I expect in GeneArt® synthetic libraries?

Three criteria are important for degenerated libraries and are quality-controlled in all GeneArt® degenerated libraries: 
  • Maximum sequence integrity of the non-degenerated parts
  • Maximum sequence variation of the degenerated positions with the requested nucleotide distribution
  • Maximum library diversity

11.  Do you offer library screening services?

We are aware that many labs are not equipped to carry out high-throughput library screenings. We offer several systems to identify the best candidates of a library. For specific information, please inquire.

12. What information do you need in order to provide a price estimate?

For GeneArt® mutagenesis services, we will need the DNA or amino acid sequence of the gene you would like to mutagenize, the host organism you plan to use (this is important for gene optimization if you choose to include it with your request), the restriction sites you need at the 5’/3’ ends and/or to avoid internally, and whether or not you want any other added motifs (e.g., Kozak sequence, stop codons, etc.)

13. Describe the details of your site-directed mutagenesis services.

Variants may contain up to four of the following modifications:
  • 5'/3' deletion of any length with additional 52 nt of new sequence on each side
  • 5'/3' modification of maximum 52 nt on each side
  • 5'/3' extension of maximum 52 nt on each side
  • Internal modifications of up to 40 nt (a maximum of 3 of these internal modification blocks are allowed)
  • Internal deletions of any length
  • Modifications (whether internal or 5´/3´) have to be separated by at least 100 bp