DNA sequencing refers to the methods and technologies that used to determine the orders of nucleotide bases in a DNA molecule, namely adenine (A), guanine (G), cytosine (C) and thymine (T). DNA sequencing enables us to perform a thorough analysis of DNA because it provides us with the most basic information of all: the sequence of nucleotides. The knowledge of DNA sequences has formed the basis of basic biological researches and clinical genetic diagnosis. There are also numerous applied technology fields such as biotechnology, forensic science and biological systematics that are heavily dependent on the information generated through DNA sequencing.
The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the human genome, in the Human Genome Project. Related projects, often by scientific collaboration across continents, have generated the complete DNA sequences the genomes of many animals, plants, and microorganisms.
The Sanger method, also referred to as dideoxynucleotide sequencing or chain termination sequencing, is based on the use of dideoxynucleotide (ddNTP) in addition to the normal nucleotides (dNTP) found in DNA. Dideoxynucleotide are essentially the same as nucleotides except they contain a hydrogen group on the 3’ carbon instead of a hydroxyl group (OH). These modified nucleotides, when integrated into a DNA sequence, prevent the addition of further nucleotides thus stop the elongation of the DNA chain. This occurs because a phosphodiester bond cannot form between the dideoxynucleotide and the next incoming nucleotide, and thus the DNA chain is terminated.
Although the various reagents, equipment and methodological strategies for carrying out DNA sequencing have undergone constant evolution to improve the simplicity, speed and reliability of the process, the basic procedure which all modern gel-based enzymatic DNA sequencing utilizes has not changed over the decades since its invention. Following steps are the outlines of the procedures of the Sanger methods:
- The region of DNA to be sequenced is amplified in some way and then denatured to produce single stranded DNA.
- A sequencing primer is annealed to the single stranded DNA.
- Dideoxynucleotide chain termination DNA sequencing then takes advantage of the fact that a growing chain of nucleotides, extending in the 5’ to 3’ direction, will terminate if, instead of a conventional deoxynucleotide, a 2’3’ dideoxynucleotide becomes incorporated. By performing four separate reactions, each containing a DNA polymerase and a small amount of one of the four dideoxynucleotides in addition to all four deoxynucleotides, four separate sets of chain-terminated fragments can be produced.
- Following the replication/termination step, these chain terminated fragments will remain bound to the single stranded DNA molecule which has acted as a template. By heating these partially double stranded molecules and adding a denaturing agent such as formamide, the single stranded chain termination molecules can be released from their template and separated using high resolution denaturing gel electrophoresis.
- The sequence of the original region of DNA is then finally deduced by examining the relative positions of the dideoxynucleotide chain termination products in the four lanes of the denaturing gel.