Sequencing strategy

Example 3. The last example covers the biggest peptide obtained from a tryptic digestion of an unknown protein. Although there are better ways to identify such peptides than de novo sequencing, this example allows us to present some additional hints that can be used in sequencing strategies. 

Ansorge, W., Voss, H., and Zimmerman, J., eds. (1997) DNA Sequencing Strategies -Automated and Advanced Approaches, Wiley, New York... 

Second large-scale sequencing strategy meeting held in Bermuda. 

S. Goletz, B. Thiede, F.-G. Hanisch, M. Schultz, J. Peter-Katalinic, S. Muiller, O. Seitz, and U. Karsten, A sequencing strategy for the localization of O-glycosylation sites of MUC1 tandem repeats by PSD-MALDI mass spectrometry, Glycobiology, 7 (1997) 881-896. 

FIGURE 3 Sequencing strategy in Applied Biosystems SOLiD platform. 

The sequencing strategy follows the order 1) preparation of pure protein, 2) selective cleavage of peptide bond 3) isolation of cleaved peptide fragments, 4) analysis of primary structure on amino acid sequencer, 5) determination of entire primary structure. Although this order has not changed since our primary structure determination work on AspAT carried out in early 1970, many improvements have been made, e.g. preparation of micro scale amount of sample and introduction of high sensitivity analytical instruments. These improvements have shortened analysis time and lowered the sample amount of required. 

In summary there is no doubt that this represents a powerful and convenient sequencing strategy of general applicability. Where the decision is to use a primed-synthesis method and the phage M-13 cloning approach (described in the next chapter) is to be avoided for any reason, the exolll procedure is likely to be the method of choice. A summarized procedure for chain terminator sequencing is given at the end of Chapter 4. 

Table II. Summary of peptide recoveries using several internal amino acid sequencing strategies...

AF, Hendrickson CL, Kieleczawa J et al (2011) Overview of DNA sequencing strategies. Curr Protoc Mol Biol. Chapter 7 Unit7.1. 

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