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390 A • ANALYTICAL CHEMISTRY, VOL. 58, NO. 3, MARCH 1986
1950s and has improved over the past few decades. The method of analysis involves the sequential degradation of one amino acid at a time from the amino terminus of the protein and its identification, usually by HPLC, as the phenylthiohydantoin derivative. Improvements in the chemistry, sepa rations, and detection methods have now resulted in the routine sequencing of many materials at the nanomole level and even lower in some cases. However, as the technology has im proved, other problems have devel oped, and the current limit to the se quencing of proteins and peptides is usually the purity of the sample, rea gents, and chemicals. Protein sequenc ing has become a truly microanalytical technique that requires careful atten tion to many details if it is to be suc cessful. Quantitative sequencing and reproducibility are presently goals, not realities, in most labs, and im provements will be necessary especial ly as sequencing is required for proc ess quality control. DNA sequencing, on the other hand, is an analytical technique that is fairly routine in many biology labora tories and can be done with a mini mum of equipment and skill with rea sonable success. The sequencing is done by either the Maxam-Gilbert chemical degradation technique (espe cially for shorter pieces of oligonucleo tides) or the Sanger method, as illus trated in Figure 2. The latter method requires the synthesis of a primer with a complementary sequence to an ini tial portion of the unknown DNA and then relies on the formation of various lengths of labeled DNA that are com plementary in sequence to the un known DNA. These various lengths are separated and analyzed on a gel, where the sequence can be read. Often from 300 to 500 bases can be deter mined in one set of reactions and gel separations. This can be a very rapid technique (sequencing a piece of DNA 1000 bases long could be done in less than one week by a skilled operator), but the length and total number of pieces of DNA to be sequenced still offer a number of challenges. In particular, the methodology is labor intensive and needs to be automated. There are a number of groups currently working on automated DNA-sequencing meth ods, and a major opportunity exists for someone who develops a useful and analytically reliable instrument. Be cause protein sequences can be de rived from the sequence of the corre sponding DNA that codes for that protein, the proper strategy of both DNA and protein sequencing can be of great use in solving the primary-struc ture problems facing many in the bio technology field.
