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Further characterization of cloned regions

©2000 written by Gary Roberts, edited by Timothy Paustian, University of Wisconins-Madison

IX B. FURTHER CHARACTERIZATION OF THE CLONED REGION IX B1. GENERATION OF A RESTRICTION MAP

Let us assume that you have generated a pool of plasmids or specialized phage by in vivoor in vitro manipulations and used a selection or screen to identify one that carries the region of interest. Just as the genetic analysis uses mutations as benchmarks, physical mapping uses sites of restriction enzymes. In each case the benchmark is actually a potential tool in the analysis. The goal is to generate a map of sites of known physical location. For most purposes a site at least every several hundred base pairs is quite sufficient. In practice, one takes the purified piece of DNA, exposes it to different restriction enzymes (one at a time) and allows their digestion of appropriate sites to go to completion. The various aliquots of DNA are then loaded onto an agarose gel and exposed to a low current. The negatively charged DNA moves through the gel toward the positive electrode, with smaller fragments moving through the gel matrix faster than larger ones. After an appropriate time, the position of the DNA "bands" is determined (e.g. by staining the DNA with ethidium bromide). By comparing the electrophoretic location of pieces of DNA of known size to that of your newly generated "restriction fragments", the size of each of the latter can be determined. The order of sites is determined through a comparison of this data with similar data from DNA which has been doubly-digested (digested with two restriction enzymes simultaneously or sequentially) as the following example shows:

Your 10-kb DNA fragment is cut into 4- and 6-kb pieces by EcoRI and into 2-, 3-, and 5- kb pieces by BamHI. While a variety of maps are possible at this point, the double digestion yields fragments at 1-, 2-, and 5-kb which is consistent only with the following map:

In approaching this sort of analysis, you want enzymes that cut the region of interest a "reasonable" number of times (one to approximately five) since too many sites make the size analysis difficult and the ordering impossible. Such an analysis does not yield much useful information by itself but sets up the two types of analyses in the following sections.

IX B2. CORRELATION OF THE CLONED REGION WITH A GENETIC MAP

The subclones can be analyzed for their ability to "complement" a known mutant as in section IX A1, or they can be used in mapping experiments to correlate the physical map with a genetic one. In the former case, one is demanding functionality, so that the cloned region will need to carry appropriate regions necessary for transcription. In the latter case, if the various subclones are moved into various Rec+ chromosomal mutants affected in the region of interest, recombination will generate a wild-type region whenever the cloned region includes the site affected by the chromosomal mutation. This is a sort of inverse deletion mapping and would "map" the chromosomal mutations to particular restriction fragments. It may also be appropriate to generate mutations in the cloned region and determine which ones affect the relevant phenotype, thus defining the relevant region genetically; this approach is covered in section IX.C2.

IX B3. PHYSICAL CHARACTERIZATION TO IDENTIFY THE RELEVANT REGION

If the relevant clone was identified by hybridization with either an oligo matching a portion of the protein sequence or with a DNA fragment encoding a homologous function from another organism, these same probes can be used to further delineate the position of the important region by hybridization to restriction fragments of the cloned region. For example, if your oligo was made to match a sequence from the amino terminus of the protein, it will necessarily hybridize to the 5' end of the coding region.

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