DNA sequence analysis: a general, simple and rapid method for sequencing large oligodeoxyribonucleotide fragments by mapping*

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Several electrophoretic and chromatographic systems have been investigated and compared for sequence analysis of oligodeoxyribonucleotides. Three systems were found to be useful for the separation of a series of sequential degradation products resulting from a labeled oligonucleotide: (I) 2-D electrophoresis†; (II) 2-D PEI-cellulose; and (III) 2-D homochromatography. System (III) proved generally most informative regardless of base composition and sequence. Furthermore, only in this system will the omission of an oligonucleotide in a series of oligonucleotides be self-evident from the two-dimensional map. The sequence of up to fifteen nucleotides can be determined solely by the characteristic mobility shifts of its sequential degradation products distributed on the two-dimensional map. With this method, ten nucleotides from the double-stranded region adjacent to the left-hand 3′-terminus and seven from the right-hand 3′-terminus of bacteriophage λ DNA have been sequenced. Similarly, nine nucleotides from the double-stranded region adjacent to the left-hand 3′-terminus and five nucleotides from the right-hand terminus of bacteriophage φ80 DNA have also been sequenced. The advantages and disadvantages of each separation system with respect to sequence analysis are discussed.

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