It was using this technique that Sanger and colleagues sequenced the first DNA genome, that of bacteriophage ϕX174 (or ‘PhiX’, which enjoys a position in many sequencing labs today as a positive control genome). By running the products on a polyacrylamide gel and comparing between the eight lanes, one is able to infer the position of nucleotides at each position in the covered sequence (except for those which lie within a homopolymer, i.e.
#Stitch era universal view sequence plus
The plus and minus technique used DNA polymerase to synthesize from a primer, incorporating radiolabelled nucleotides, before performing two second polymerisation reactions: a ‘plus’ reaction, in which only a single type of nucleotide is present, thus all extensions will end with that base, and a ‘minus’ reaction, in which three are used, which produces sequences up to the position before the next missing nucleotide. This technique was used in two influential yet complex protocols from the mid-1970s: Alan Coulson and Sanger's ‘plus and minus’ system in 1975 and Allan Maxam and Walter Gilbert's chemical cleavage technique. The next practical change to make a large impact was the replacement of 2-D fractionation (which often consisted of both electrophoresis and chromatography) with a single separation by polynucleotide length via electrophoresis through polyacrylamide gels, which provided much greater resolving power. However the actual determination of bases was still restricted to short stretches of DNA, and still typically involved a considerable amount of analytical chemistry and fractionation procedures. Incorporation of radioactive nucleotides could then be used to infer the order of nucleotides anywhere, not just at the end termini of bacteriophage genomes. It was not long before this principle was generalized through the use of specific oligonucleotides to prime the DNA polymerase. Making use of the observation that Enterobacteria phage λ possessed 5′ overhanging ‘cohesive’ ends, Ray Wu and Dale Kaiser used DNA polymerase to fill the ends in with radioactive nucleotides, supplying each nucleotide one at a time and measuring incorporation to deduce sequence. It was around this time that various researchers began to adapt their methods in order to sequence DNA, aided by the recent purification of bacteriophages with DNA genomes, providing an ideal source for testing new protocols. It was also by using this 2-D fractionation method that Walter Fiers' laboratory was able to produce the first complete protein-coding gene sequence in 1972, that of the coat protein of bacteriophage MS2, followed four years later by its complete genome. In parallel, Fred Sanger and colleagues developed a related technique based on the detection of radiolabelled partial-digestion fragments after two-dimensional fractionation, which allowed researchers to steadily add to the growing pool of ribosomal and transfer RNA sequences, ,. However, by combining these techniques with selective ribonuclease treatments to produce fully and partially degraded RNA fragments (and incorporating the observation that RNA contained a different nucleotide base ), in 1965 Robert Holley and colleagues were able to produce the first whole nucleic acid sequence, that of alanine tRNA from Saccharomyces cerevisiae. Despite these advantages, progress remained slow, as the techniques available to researchers – borrowed from analytical chemistry – were only able to measure nucleotide composition, and not order. Furthermore, RNase enzymes able to cut RNA chains at specific sites were already known and available. Not only could these be readily bulk-produced in culture, but they are also not complicated by a complementary strand, and are often considerably shorter than eukaryotic DNA molecules. Initial efforts focused on sequencing the most readily available populations of relatively pure RNA species, such as microbial ribosomal or transfer RNA, or the genomes of single-stranded RNA bacteriophages. Strategies developed to infer the sequence of protein chains did not seem to readily apply to nucleic acid investigations: DNA molecules were much longer and made of fewer units that were more similar to one another, making it harder to distinguish between them. However, the ability to ‘read’ or sequence DNA did not follow for some time. Watson and Crick famously solved the three-dimensional structure of DNA in 1953, working from crystallographic data produced by Rosalind Franklin and Maurice Wilkins, , which contributed to a conceptual framework for both DNA replication and encoding proteins in nucleic acids.