Two-hybrid system as an example of a "handle" for cloning

Biochemistry/MCB 568 -- Fall 2007
John W. Little--University of Arizona

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In any recombinant DNA experiment, you must have some way of identifying the clone you're interested in. Let's call this a "handle", since it's a way to take hold of the proper clone. You can't just "clone your gene"; you need a way to recognize it among many other clones, typically >10⁶ , that are not what you want. There are many ways to do this:

1. Two-hybrid system: Identifies clones containing a protein that interacts with your bait. Typically, most proteins will not interact with the bait; hence, finding one that does is a good handle.

2. Hybridization: If you have a gene that is closely related to the one you wish to clone, you can use methods similar to a Southern blot (termed a plaque lift or colony lift) to recognize your clone. For example, if you wished to clone the hemoglobin genes from a black-tailed rattlesnake, you could use hemoglobin genes from some other reptile as a probe.

3. Microsequencing of a protein: If you have a purified protein, you can obtain some amino acid sequence of the protein (there are many facilities that will do this, including one at the U of A). You don't need a lot of protein for microsequencing. Moreover, it doesn't have to be completely pure; you can run an SDS gel, transfer to a membrane, stain the proteins, and cut out the bands, which can individually be sequenced. How you use this information depends on the situation.

If your organism has been sequenced (as in the cases, for example, of E. coli, S. cerevisiae, Drosophila melanogaster, and Homo sapiens), you can quickly find your gene in the sequence. These sequences are found online.

If your organism has not been sequenced, you can use this limited sequence information to design a "degenerate" oligonucleotide, which you then use to probe as above. This oligo will contain many sequences, because the genetic code is degenerate and you need to account for all the possibilities. It is is a good idea to pick parts of the sequence coding for amino acids with 1 or 2 codons; e.g., if your sequence included Met-Trp-His-His-Lys, you would need only 8 oligos, because Met and Trp have one codon, while His and Lys have two. Oligos can be made with each position containing a desired fraction of two (or more) bases; in this case, your oligo might be ATG TGG CAY CAY AAP, where Y is pyrimidine and P is purine.