Research Update
Site-Directed Mutagenesis
Site-directed or oligonucleotide-directed mutagenesis is a key method in the study of protein structure/function and gene expression control. If the nucleotide sequence of the gene is known, an oligonucleotide can be manufactured to introduce a single base change in the codon corresponding to the specific amino acid to be altered. The oligonucleotide is then used to generate a set of clones that can be identified and propagated.
If less structural information is known, combinatorial mutagenesis can use random oligonucleotides. Use of randomers overcomes the tediousness and/or inadequacy of the first method when there is uncertainty regarding which region of the sequence to vary. The technique allows the development of a larger, more diverse library of clones, thus increasing the chances of success from a single series of experiments.
TriLink has done extensive research to perfect the synthesis of randomers. In particular, to overcome the synthesis challenges of competing species-specific characteristics that make producing a truly random oligonucleotide problematic.
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Randomer Oligonucleotides Article
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Technology Spotlight
The Trimer Oligo Solution
While randomers are often used in protein mutagenesis procedures, they have several short comings. First, it is very difficult to achieve a truly random mixture. The second and more problematic issue is the inevitable introduction of stop codons. Fortunately, the ability to direct combinatorial mutagenesis using randomized oligonucleotides has been advanced by the use of trimer oligonucleotides.
Trimer incorporated oligonucleotides have become an increasingly popular method for incorporating base variability with specific ratios of amino acid mutations. This method allows the randomization of specific sites to yield specific amino acids and to avoid stop codons and sequence repeats. Commercially available mixtures of trimer phosphoramidites provide all nineteen of the common amino acids. The 20th amino acid, the stop codon, is adroitly missing from the mix. Custom mixes of any number of amino acids at any percentage can be made and then coupled into an oligonucleotide sequence as a single incorporation. TriLink is one of the leading providers of trimer-incorporated oligonucleotides.
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Trimer phosphoramidites from Glen Research |
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Q - What is the split and pool method for introducing mutagenic sites?
A - Degeneracy can be obtained through insertions, deletions and/or point mutations, by introducing mixed sequences into your oligonucleotide. One way to accomplish this is using the “split and pool” method of oligonucleotide synthesis. This method provides a simple, albeit tedious and labor intensive approach to introducing variations by splitting out the CPG, synthesizing various determined sequence portions and then pooling the unique sequences before repeating the process. An increase in labor, decrease in coupling efficiency and quantitative limitations however have discouraged widespread adoption of this synthesis method.
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