Exercise 14.12 - Other studies

LEVEL III

Once DNA has been purified and analyzed, there are many procedures that can be followed. Hydroxyapatite columns can easily separate single stranded DNA from double stranded DNA and are used extensively in gene transplant work. This can be appended to the formation of DNA-RNA hybrids, whereby an mRNA is re-annealed to DNA, all single strands eliminated and the resulted hybrid re-separated. The remaining DNA strand is the gene for that mRNA (although not complete, some start, stop and housekeeping codes will be missing).

If one has a sufficient quantity of RNA's, it is thus possible to create a DNA "library" for these genes. Modern approaches will use the mRNA to synthesize DNA rather than isolating it through hybridizion. DNA synthesized from RNA is known as cDNA, which presents us with powerful "libraries" for analysis of cell function at the gene level.

Once obtained, the DNA can be readily sequenced, although the techniques are too lengthy for our current analysis in a general cell biology laboratory. Gene sequencing has now been automated, and the key to success is the isolation of pure unique copy DNA (that is DNA that has only one sequence to analyze). Once sequenced, computer searches can be made of known libraries to compare the gene with that from other organisms.

For an excellent series of sequencing techniques cf. Methods of DNA and RNA Sequencing, ed. S.M. Weissman. Praeger Publishers, New York. 1983, or Current Procols in MOlecular Biology edited by F.M Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.A. Smith, J.G. Seidman and K.Struhl. Greene Publishing Associates, Wiley Interscience, Brooklyn, NY, 1988.

Further analyses of RNA are also possible. Affinity chromatography allows the separation of mRNA, which can then be analyzed for specific base sequence. This mRNA could be used for translation on isolated ribosomes, or used to synthetically produced DNA copies (cDNA).

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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- cellab@gac.edu