Determining the sequence of bases of a given DNA molecule in a reasonable period of time was largely an unapproachable task until the mid 1970s, when two very different methods for accomplishing this goal were described. Without the development of a rapid means to sequence DNA, a majority of the dramatic developments in scientific research today would have never been realized.
Chemical DNA Sequencing
In 1977 Allan Maxam and Walter Gilbert of Harvard University published a paper describing a chemical means to determine the sequence of bases in a molecule of DNA (1). In this method, a purified strand of DNA had to have a radioactive label attached to one end of the strand. The labeled DNA sample was divided into equal fractions and they were then subjected to various chemical reactions.
Each combination of chemicals would induce the breakage of the DNA strand at specific places corresponding to the type of base (nucleotide) that was present. There were four different sets of reactions, one for each nucleotide, that were used on the DNA and the reactions were specifically meant to be incomplete. This would leave a collection of fragments in any given reaction that were cleaved at a position in the chain corresponding to a particular nucleotide. The sample would thus have a collection of fragments of different length, but all of which were cleaved adjacent to a specific base.
The fragmented DNA samples were separated on high resolution, denaturing polyacrylamide gels. These gels could separate DNA fragments that were different by a single nucleotide in length. After exposing the radioactive fragments to a piece of film, the film would be developed showing a series of staircases of bands. By running the different reaction products immediately adjacent to each other on the same gel, the labeled fragments would be run so that the sequence of bases could be read by following the bands up the gel.
Unfortunately, the chemical method proved to be more cumbersome and used more dangerous chemicals than another method developed at the same time. And this other method, the dideoxy chain termination method, has served as the backbone for future developments in rapid DNA sequencing.
Sequencing DNA by Synthesis
The ability to rapidly sequence DNA really came into being with the development of the dideoxynucleotide chain termination method (2). Sanger and colleagues developed a method where DNA to be sequenced was enzymatically copied starting at a specific point using a DNA polymerase enzyme. This was done in the presence of mixtures of nucleotide precursors where mixed in the reactions were lower concentrations of similar nucleotides that had a chemical modification that, once incorporated, made it impossible to extend the newly synthesized chain any further. These special nucleotides were known as dideoxynucleotides because they were missing an important hydroxyl group at one position in the molecule. This chemical change would stop the synthesis of the new chain wherever it was incorporated.
By setting up four different synthesis reactions, one for ‘A’, one for ‘T’, one for ‘G’, and one for ‘C’, the specific reactions had a small amount of the corresponding dideoxy base added. For reading ‘A’, dideoxy A was added, for ‘T’ dideoxy T was added and so on. By carefully controlling the concentrations of the reactants, a given reaction would produce a mixture of partially completed strands, all of which would stop at positions along the chain where the dideoxynucleotide was incorporated. By running the four different reactions alongside each other on the same type of high resolution gel as used for the Maxam and Gilbert reactions above, a staircase of bands could be “read” up the gel.
Originally performed using radioactive nucleotides and film images, this method has been performed for many years now with fluorescent nucleotides and specialized machines that can detect the molecular fluorescence as the samples migrate through the gel.
Modern DNA Sequencing Methods
Today, many facilities use the so-called “Sanger” method to sequence DNA. But for many, the samples are now run through high throughput capillary electrophoresis machines and no polyacrylamide gels are used. And recently developed “next generation” sequencing methods, which promise to deliver tens of thousands of bases of DNA sequence per experiment, don’t even use the same methods anymore. But for the last 30 years, variants of the dideoxy chain termination method have, for many research labs, served as the workhorse method for determining the sequence of bases in a DNA molecule.
(1) Maxam AM, Gilbert W. (1977) A new method for sequencing DNA. Proc Natl Acad Sci U S A.; 74(2):560-564.
(2) Sanger F, Nicklen S, Coulson AR. (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A.; 74(12):5463-5467.
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