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

Abstract

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 electrophoresisdagger; (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 lambda 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 phi80 DNA have also been sequenced. The advantages and disadvantages of each separation system with respect to sequence analysis are discussed.

The nucleotide sequence of the intergenic region between the 5.8S and 26S rRNA genes of the yeast ribosomal RNA operon. Possible implications for the interaction between 5.8S and 26S rRNA and the processing of the primary transcript

“…We obtained the same product in a nearly unimolar yield upon ribonuclease Ti digestion of S. carlsbergensis 26S rRNA and we found pU-U-G-Ap upon digestion with ribonuclease U2 of kethoxal modified RNA (this modification blocks the G-residues; data not shown). In order to extend this sequence we applied the "wandering spot" method [16] to [5' 32P] Fig. 4 [23].…”

Increase in nucleolytic activity of ribonuclease T1 by substitution of tryptophan 45 for tyrosine 45

“…The protein concentrations were determined by Lowry's method or, in the case of the purified mutant enzyme, by using a modified molar absorption coefficient (&278 = 25330 M-' cm-') which was derived from those of RNase TI (~2 7 8 = 21170 M-' cm-'), tryptophan (&278 = 5500 M-' cm-') and tyrosine = 1340 M-' cm-'). cellulose TLC (CEL 300 DEAE/HR-2/15, Machery-Nagel) by using partially hydrolyzed RNA solution (homomixture VI [13], as solvent system and detected by autoradiogram (homochromatography [14]). pGp is not separated from pG > p in this system.…”

Complementarity Between the 5'- and 3'-terminal Sequences of Rice Stripe Virus RNAs

“…The second dimension was homochromatography as described previously (Jay et al, 1974;Wengler & Wengler, 1981). RNA sequencing.…”