Quantitative analysis of DNA-sequencing electrophoresis.
At the heart of the DNA-sequencing process is a remarkably selective electrophoretic separation of up to 1000 oligonucleotide fragments, each differing in size by only a single nucleotide unit. A quantitative analysis of this separation is performed in terms of both selectivity and efficiency. It is shown that both the Ogston sieving and reptation migration mechanisms are operative. It is demonstrated that, under the conditions used in traditional sequencing electrophoresis, Joule heating does not significantly contribute to band broadening, and that diffusion is the primary contributor to plate height. An analytic expression is derived relating the peak width for each fragment to its molecular size. Calculations are presented showing that, when longer sequences are required, the maximum electrical field strength will be limited by the influence of biased reptation on the separation selectivity. Finally, it is shown that, when short sequences are required, the electrical field strength is limited by the ability to dissipate Joule heat, and that in these cases a tube format will be approximately 50% faster than a slab having a thickness equivalent to the tube diameter.
Grossman
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Applied Biosystems, Inc., Foster City, California 94404.
Menchen
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Hershey
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D004247:DNA / Q000032:analysis* / Q000737:chemistry; D004058:Diffusion; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D006358:Hot Temperature; D008956:Models, Chemical; D015394:Molecular Structure; D009710:Nucleotide Mapping; D009838:Oligodeoxyribonucleotides / Q000032:analysis
D009838:Oligodeoxyribonucleotides; D004247:DNA