In a previous article on the Human Genome Project, we talked about the feat of obtaining the complete sequence of our genome, as well as its applications. For this, it was necessary to determine the order of the nucleotides that make up DNA , which required a considerable effort on the part of several universities and research centers over a decade. In this article we will explain which was the first technique developed that allowed the sequencing of genes, the Sanger method and which today is still the most accurate.
Basic notions about DNA
Let’s first review some general notions. The genome is the set of genes of an organism or species. These are encoded in deoxyribonucleic acid (DNA), which is made up of four types of subunits, called nitrogenous bases. The nitrogenous bases of DNA are adenine, thymine, cytosine, and guanine. Each DNA sequence that codes for a protein are called a gene.
DNA is a double strand in the form of a helix, on each side of the chain there are two complementary nitrogenous bases: adenine is complementary to thymine and cytosine is complementary to guanine. Therefore, if we know that at a point in one of the two chains there is a cytosine, we know that at that same point in the complementary chain there is guanine. This is important to understand the sequencing method developed by Sanger.
Frederick Sanger was a British biochemist, who received the 1958 Nobel Prize in Chemistry for his research on the structure of proteins, and in 1980 he received a second Nobel Prize in Chemistry for developing the first DNA sequencing technique. His findings on DNA sequencing were published in 1975 and marked the beginning of numerous genetic investigations.
The Sanger sequencing method started from a single DNA strand, which would have previously been separated from its complementary strand. The DNA synthesis process is carried out by the polymerase enzyme (which we talk about in this article about PCR tests ), which is added to the DNA strand that you want to sequence. Polymerase, in the presence of a DNA strand, a sequence from which synthesis starts (called “primer”) and nitrogenous bases, is capable of synthesizing the complementary DNA strand.
¿ How to obtain the sequence of the DNA strand being synthesized? By stopping DNA synthesis so that we know what the last nucleotide was added to the chain, and putting the many incomplete chains of different lengths in order.
For this, DNA synthesis is carried out in four separate reactions. To each of them are added the four nucleotides, and a dideoxynucleotide (adenine, thymine, cytosine, or guanine). Dideoxynucleotides lack a molecule that is necessary for DNA synthesis to continue so that when one is added to the chain, synthesis stops and the strand remains incomplete.
Sanger sequencing method