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DNA sequencing with direct blotting electrophoresis.

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PMC
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  • Research Article

Abstract

A method for transferring the DNA molecules of sequencing reaction mixtures onto an immobilizing matrix during electrophoresis has been developed. A blotting membrane moves with constant speed across the end of a very short, denaturing gel and collects the molecules according to size. A constant distance between bands for molecules differing in length by one nucleotide is obtained over a large range (approximately 600 nucleotides with a 5% gel), simplifying the determination of DNA sequences considerably. Reliable sequences of 500 nucleotides can be read and sequence features up to greater than 1000 nucleotides are revealed in a single experiment. The sequencing of a potential Z-DNA-forming fragment from Escherichia coli DNA is given as an example and possible further developments are discussed.

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