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About genomes (glossary)

Initial stage of DNA sequencing technologies

Sequencing of nucleic acids started in the mid-1960s with the sequencing of low-molecular weight RNAs such as tRNA. At that time, sequencing was performed using base-specific cleavage by ribonucleases, combined with techniques for separating and isolating nucleic acids, such as chromatography and electrophoresis. Later, when methods for labeling with radioactive isotopes were introduced, it became possible to perform high-speed sequencing using a small amount of a sample.

Unlike the case for RNA sequencing, the development of DNA sequencing technologies took a long time, as no enzyme with base-specific cleavage characteristics similar to that for ribonucleases was available. Thus, for long DNA molecules, the DNA was first transcribed to RNA using an RNA polymerase, and then the RNA molecule was sequenced. In the case of short DNA fragments, the sequencing could be performed using a special separation technology in which electrophoresis was combined with chromatography. However, it was only possible to read 10−20 bp using this technique.

The situation was completely changed when restriction enzymes that could sequence-specifically degrade a DNA molecule were found and in vitro recombinant DNA technology subsequently developed. In 1977, a DNA sequencing technology called the Maxam–Gilbert method was developed, in which end-labeling of a DNA molecule, base-specific chemical cleavage, and denaturing gradient gel electrophoresis were combined. Together with in vitro recombinant DNA technology, the Maxam–Gilbert method converted molecular biology into a true science of molecules. At that time, the impact of the Maxam–Gilbert method on biologists was huge. By the time a paper written by A. M. Maxam and W. Gilbert describing their method had been published, knowledge of the method had already spread all over the world.

However, the Maxam–Gilbert method had two disadvantages: it used a large amount of a radioisotope for labeling DNA molecules, and the reagent used for chemical cleavage was highly poisonous and unstable. To overcome these disadvantages, the Sanger method was developed, which used DNA polymerases and dideoxynucleotides. Later, the Sanger method became the basic technology for automatic DNA sequencers. Like the Maxam–Gilbert method, the Sanger method was published in 1977. However, widespread use of the Sanger method only started after dideoxynucleotides became commercially available.