SEQUENCES IN NUCLEIC ACIDS

Determination of nucleotide sequences in RNA has been facilitated by the development of small-scale techniques for fractionating 32P-labelled oligonucleotides produced by digestion with ribonucleases, particularly ribonuclease T1. Two-dimensional fractionations on modified papers and thin layers by ionophoresis and chromatography give "fingerprints", which may be used to characterise a given RNA and to isolate pure oligonucleotides as a first step in sequence analysis. Micro methods have been developed for determining the composition and sequence of the isolated oligonucleotides. These techniques have made it possible to determine the complete sequences of RNAs up to about 180 nucleotides long and to deduce a large part of the sequence of an RNA bacteriophage (3300 nucleotides long). Progress in the determination of sequences in DNA has been slower than in RNA due to the large size of the simplest DNA molecules and the lack of suitable deoxyribonucleases for specific degradation. However the small-scale techniques have recently been extended to the study of 32p-labe lied DNA and it has been possible to determine two sequences of about 50 residues from bacteriophage øX. The methods used and other possible approaches to DNA sequencing will be discussed. Another method has been developed in which a specific region in a DNA molecule is sequenced by a copying procedure using DNA polymerase primed by a specific oligonucleotide. In this way a sequence of 50 residues was deduced in bacteriophage f1 DNA.