Sequencing chemistries

George S M, Sneh O, Diiion A C, Wise M L, Ott A W, Okada L A and Way J D 1994 Atomic iayer controiied deposition of SiO, and Ai,0, using ABAB.. . binary reaction sequence chemistry Appl. Surf. Sc/. 82/83 460-7... 

Ott A W, McCariey K C, Kiaus J W, Way J D and George S M 1996 Atomic iayer controiied deposition of Ai203 fiims using binary reaction sequence chemistry App/. Surf. Sc/. 106 128-36... 

Ott A W, Kiaus J W, Johnson J M and George S M 1997 Ai203 thin fiim growth on Si(IOO) using binary reaction sequence chemistry Thin Solid Films 292 135-44... 

Sequencing chemistry Reversible terminator Pyrosequencing Ligation Proton detection... 

The DNA sequencing chemistry begins with a base-modification reaction, the extent of which determines the frequency of DNA cleavage in the subsequent phosphate-elimination reaction. The number of bases modified in each molecule depends on the concentration of dimethylsulphate (G and G A reactions) and hydrazine (C T reactions) as well as the temperature and duration of the reaction. For speed and convenience the Maxam-Gilbert procedure makes use of temperature shifts and dilution to control the rate and extent of these reactions. The reagents are mixed at 0°C, incubated at 20°C for the required time and the DNA precipitated with cold sodium acetate and ethanol to slow down or halt the reaction. A fixed concentration of the different reagents is usually used so the main factor determining the extent of reaction is the time of incubation at 20°C. 

Figure 2 A ladder sequence through 6 cycles of a synthetic peptide (CD28-3PY) carried out on a total of 17.5 picomoles of starting peptide. The peptide is especially interesting in that it contains both a proline and phosphorylated tyrosine residue. Both residues undergo the sequencing chemistry satisfactorily.

Superoxide dismutase, an N-terminally blocked protein, was applied to a Zitex reaction membrane (approximately 1 nmol) in 10 xl of 1% aqueous trifluoroacetic acid. The first three cycles of C-terminal sequence analysis resulted in the identification of Lys (K) cycle-1, Ala (A) cycle-2, and lie (I) cycle-3 (Figure 3). The chemical background remained relatively stable as a thiohydantoin background increased, in part, attributed to internal cleavages as analogously observed for amino-terminal sequencing chemistry. 

Scheme 1, Perkin-Elmer C-terminal sequencing chemistry...

Automation of the C-Terminal Sequencing Chemistry. The instrument used for automation of the chemistry described in this manuscript has been described previously (5). 

Chessari, S. Thomas, R. Polticelli, F. Luisi, P. L., The production of de novo folded proteins by a stepwise chain elongation A model for prebiotic chemical evolution of macromolecular sequences. Chemistry Biodiversity 2006, 5, 1202-1210. 

One thing to note is that the field has moved on since the genome has been sequenced. Chemistry is still the rate-limiting step. In other words, new chemical structures that have unique activities are still hard to come by. 

What changes in the sequencing technology will allow such improvements There are two key aspects to the problem these are accuracy, and throughput. Accuracy is determined by both the quality of the raw data, and by the ability of the software to analyze the raw data effectively to yield the DNA sequence. At this time, there is room for improvement in both aspects our current work is focused upon improving the sequencing chemistry itself, and the analysis software is rapidly evolving in work performed at Applied Biosystems. 

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