In Vitro Compartmentalization - Method - Breaking Emulsion and Coupling of Genotype and Phenotype

Breaking Emulsion and Coupling of Genotype and Phenotype

Once transcription and/or translation has completed in the droplets, emulsion will be broken by successive steps of removing mineral oil and surfactants to allow for subsequent selection. At this stage, it is crucial to have a method to ‘track’ each gene products to the encoding gene as they become free floating in a heterogeneous population of molecules. There are three major approaches to track down each phenotype to its genotype. The first method is to attach each DNA molecule with a biotin group and an additional coding sequence for streptavidin (STABLE display). All the newly formed proteins/peptides will be in fusion with streptavidin molecules and bind to their biotinylated coding sequence. An improved version attached two biotin molecules to the ends of a DNA molecule to increase the avidity between DNA molecule and streptavidin-fused peptides, and used a low GC content synthetic streptavidin gene to increase efficiency and specificity during PCR amplification. The second method is to covalently link DNA and protein. Two strategies have been demonstrated. The first is to form M.HaeIII fusion proteins. Each expressed protein/polypeptide will be in fusion with Hae III DNA methyltransferase domain, which is able to bind covalently to DNA fragments containing the sequence 5’-GGC*-3’, where C* is 5-fluoro-2 deoxycytidine. The second strategy is to use monomeric mutant of VirD2 enzyme. When a protein/peptide is expressed in fusion with Agrobacterium protein VirD2, it will bind to its DNA coding sequence that has a single-stranded overhang comprising VirD2 T-border recognition sequences. The third method is to link phenotype and genotype via beads. The beads used will be coated with streptavidin to allow for the binding of biotinylated DNA, in addition, the beads will also display cognate binding partner to the affinity tag that will be expressed in fusion with the protein/peptide.

Read more about this topic:  In Vitro Compartmentalization, Method

Famous quotes containing the words breaking and/or coupling:

    Freedom is poetry, taking liberties with words, breaking the rules of normal speech, violating common sense. Freedom is violence.
    Norman O. Brown (b. 1913)

    The time of the seasons and the constellations
    The time of milking and the time of harvest
    The time of the coupling of man and woman
    And that of beasts. Feet rising and falling.
    Eating and drinking. Dung and death.
    —T.S. (Thomas Stearns)