DNA sequencing is the technique of determining the sequence of bases within a DNA molecule. Its development has significantly advanced our knowledge of genetics.
Using the Sanger sequencing technique, scientists were able to decipher the genetic code for the first time. It follows the natural process of DNA replication.
The double helix of DNA is "unzipped" by enzymes.
Once unzipped, the two split DNA strands serve as templates for the creation of two more DNA strands.
A fragment of RNA known as a "primer" attaches to the template strand.
DNA polymerase attaches itself to the primer.
DNA polymerase initiates the synthesis of a new strand of DNA by integrating complementary free nucleotide bases (A, C, G, and T) from the template strand.
This procedure is repeated until two identical duplicates of the initial double-stranded molecule are created.
To separate the two strands of DNA, the double helix is "denatured" (broken down) by heat or chemicals. These will then serve as DNA synthesis templates.
Addition of a primer, DNA polymerase, and nucleotide bases (A, C, G, and T). One or more of these nucleotides are radioactively labelled in order to detect newly synthesised DNA.
Other variants of these bases, called as terminators, are also added in minute quantities, beginning with the 'A' terminator. Terminators inhibit DNA replication. Therefore, the 'A' terminator will halt DNA synthesis when a 'A' base is introduced (the 'C' terminator will halt DNA synthesis when a 'C' base is added, etc.).
This results in a combination of radioactive DNA fragments of varying lengths that all end in the same base, in this case A.
The four distinct reactions are then put onto separate lanes of an acrylamide gel, and the DNA fragments are sorted by size using electrophoresis.
Initially, they are loaded into distinct lanes of a gel (like a slab of firm, transparent jelly). At each end of the gel, electrodes are put and an electrical current is applied.
DNA is negatively charged, therefore when an electric current is supplied, DNA fragments migrate to the positively charged end of the gel (opposites attract!).
Smaller pieces pass through the gel more rapidly than larger ones.
The radioactively labelled DNA is subsequently visualised using X-ray film exposed to the gel. The DNA that has been radioactively tagged will render the film black. This film is known as an autoradiogram.
Each band in the film refers to a specific terminator version being added to one of the bases (A, C, G or T). Therefore, the DNA sequence may be read from the bottom of the film.