USE OF POLYNUCLEOTIDE KINASE IN FINGERPRINTING NON-RADIOACTIVE NUCLEIC ACIDS

A method is described for obtaining fingerprints from non-radioactive nucleic acids by labelling a digest with the aid of 5′-hydroxyl polynucleotide kinase. The nucleotides thus obtained are labelled in the 5′-phosphate and can be fractionated by two-dimensional ionophoresis. The technique has been applied to depurinated fd DNA and to RNase T1 digests of formylmethionine tRNA. Several pyrimidine sequences in fd DNA have been established, using phosphodiesterase digestion for further analysis of oligonucleotides. The same procedure can be applied to smaller nucleotides in the RNA digest. Sequence determination of RNA is, however, complicated by some side reactions accompanying phosphorylation of the digest. © 1969.