Ernest Jay

A rellable mapping method for sequence determination of oligodeoxyribonucleotides by mobility shift analysis

Abstract The method for sequence analysis of large oligodeoxyribonucleotides based on the characteristic mobility shifts of their sequential partial degradation products on two-dimensional homochromatography has been perfected using a large number of synthetic oligodeoxyribonucleotides of defined sequences as standards. Flat bed electrophoresis with careful temperature control gave entirely reproducible mobilities in the first dimension. Using this information, an accurate formula has been derived for calculating the relative electrophoretic mobilities of oligodeoxyribonucleotides of any composition. This formula is used to calculate the mobility shifts between two consecutive oligodeoxyribonucleotides in a series of partial products of an unknown oligomer distributed in the two-dimensional homochromatogram which differ by one nucleotide in length. This is compared with the observed mobility shift value to identify the added nucleotide. This provides a direct and rapid method for obtaining the unambiguous sequence of an entire oligodeoxyribonucleotide up to 15 nucleotides in length.

Nucleotide sequence with elements of an unusual two-fold rotational symmetry in the region of origin of replication of SV40 DNA☆

Abstract We have determined the nucleotide sequence at or near the origin of replication of simian virus 40 DNA. This 28-nucleotide-long sequence contains two 8-nucleotide-stretches, complementary to each other, located two nucleotides away from each side of the axis of symmetry. Within each of the 8-nucleotide-stretch is a minor axis of symmetry with 4-nucleotides on each side. This highly symmetrical sequence may be of considerable importance for recognition by a biologically important protein molecule.

Nucleotide sequence analysis of DNA: XI. The 3′ terminal sequences of bacteriophage λ and φ80 DNA

Two procedures have been developed and applied to the determination of the 3′ terminal sequences of λ DNA and φ80 DNA. In the first procedure, each 3′ terminus was specifically labeled with a single 32P-nucleotide. Radioactive oligonucleotides of different lengths were obtained by partial pancreatic deoxyribonuclease digestion. From the characteristic mobilities of these oligonucleotides in two dimensional fractionation systems, the 3′ terminal sequence -ACCCGCG for the r-strand and -GGTTACG for the l-strand of λ DNA have been determined. In the second procedure, approximately six nucleotides were removed from each 3′ terminus with exonuclease III, and they were replaced with radioactive nucleotides by partial repair synthesis. After enzymatic digestion and sequence analysis, the above sequences have been confirmed. The 3′ terminal sequences in φ80 DNA are identical to those in λ DNA at least up to the fifth nucleotide from the 3′ ends.