Application of Fingerprinting Techniques to Iodinated Nucleic Acids

Abstract

Several techniques of RNA and DNA fingerprinting and determination of sequence have been applied to nucleic acids labeled with 125I. Fingerprints of human 5S RNA and bacteriophage f2 RNA resemble those of their noniodinated counterparts both in complexity and in specific pattern. Iodination as used here is thus a general labeling procedure, and appears principally to label cytidine residues. This iodination method shows little sensitivity to potential structure in single-stranded RNA molecules, yields stable oligonucleotide products in a reproducible manner, and does not change the specificity of several ribonucleases and deoxyribonucleases.Recent advances in the techniques of nucleic acid fingerprinting and determination of sequence have yielded biologically interesting RNA and DNA sequences (1-5) and have fostered comparative studies of closely related nucleic acid species (6, 7). Previous experiments concentrated upon nucleic acids that can be labeled to high specific activity with 32p and isolated in high yield, usually from microorganisms.With increasing interest in applications of these methods to eukaryotes, indirect approaches to obtain radioactive nucleic acids are being studied. For example, one can attempt to copy isolated nucleic acids with various polymerases, obtaining complementary copies of high specific radioactivity (8-10). Alternatively, nonradioactive nucleic acids are labeled after isolation from the organism (11).Several reports have appeared describing methods for iodination of nucleic acids with 125I (12, 13). Prensky et al. (13) have described a modification of Commerford's iodination procedure (12) by which they obtain RNA of very high specific radioactivity (106-101 dpm/,g).

Evidence for the protection of specific RNA sequences in globin messenger ribonucleoprotein particles

“…This fingerprint is characterized by the numbered oligoribonucleotides depicted in the figure, and it is very similar to that obtained by ROBERTSON et al for iodinated duck globin mRNA (24). We recall that this fingerprint shows exclusively the cytosine containing oligoribonucleotides from ca and B duck globin chains.…”

Nucleotide Sequences of Human Globin Messenger Rna *

“…The mobilities of some of the spots differ somewhat in the two fingerprints, making exact matching occasionally equivocal. Differences in mobility in the first dimension may be caused by the modification of the cytosine residues by 1251 (37). Nevertheless, the overall patterns are unequivocally quite similar.…”

“…Intact pure 10S RNA was labeled with 125I by the method of Commerford (25). These techniques have been used by others to label globin mRNA for fingerprint analysis (24,37).…”

Translation of satellite tobacco necrosis virus ribonucleic acid by an in vitro system from wheat germ

“…As seen in Table II, if containing a 3'-terminal Cp. Both of these specific oligonucleotides would contain l25I due to the cytidine-specific iodination procedure used to make the original STNV [l25I]RNA (Robertson et al, 1973b). Thus, these two l25Ilabeled oligonucleotides should be among the few 125I-labeled oligonucleotides detected in a ribonuclease A digest of ,25Ilabeled, previously ribosome protected, mRNA fragment.…”