Linking Genotype and Phenotype

In vitro methods have the advantage that the size of the library to be screened can be as large as 1012-1013, whereas the size achievable using expression into cells (in vivo methods) have between 108 and 1010 different DNA members. Proteins that interfere or are detrimental to metabolic cell processes (toxins, inhibitors of protein synthesis) cannot be selected using in vivo methods.13 

In vivo methods for creating genotype-phenotype linkages are well established and exploit the cellular machinery necessary to process some proteins. Cell surface display of proteins coupled with fast cell-sorting systems can increase the size of the library that can be displayed and selected or screened. 

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Nourian, Z., Danelon, C. Linking genotype and phenotype in protein synthesizing liposomes with external supply of resources. ACS Synth. Biol. 2,186-193 (2013)... 

O. Fiehn, Metabolomics The link between genotype and phenotype. Plant Mol. Biol. 78,155 171 (2002). 

Fiehn, O., Metabolomics — the link between genotypes and phenotypes, Plant Mol. Biol., 48, 155, 2002. 

In ribosome display, the physical link between genotype and phenotype is accomplished by mRNA-ribosome—protein complexes, which are directly used for selection. If a library of different mRNA molecules is translated, a protein library results in which each protein is produced from its own mRNA and remains connected to it. Since these complexes of the proteins and their encoding mRNAs are stable for several days under the appropriate conditions, very stringent selections can be performed. As all steps of ribosome display are carried out in vitro, reaction conditions of the individual steps can be tailored to the requirements of the protein species investigated, as well as the objectives of the selection or evolution experiment. Application of ribosome display has produced scFv fragments of antibodies with affinities in the picomolar range from libraries prepared from immunized mice (Hanes et al., 1998) and more recently from a naive, completely synthetic library (Hanes et al., 2000), and has been used to evolve improved off-rates and stability (Jermutus et al., 2000). 

Selection and Methods to Link Genotype with Phenotype... 

Fig. 2. (opposite page) The ABCBl gene page lists the alternate gene names, features the PharmGKB primary genotype and phenotype data in the center, literature data below, and links to external sites in the right hand panel. 

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