Where did DNA sequencing begin?
1970s: Sanger sequencing method
- The DNA double helix is ‘unzipped’ by enzymes.
- Once unzipped, the two separated strands act as templates for creating two more strands of DNA.
- A short piece of RNA called a ‘primer’ binds to the template strand.
- An enzyme called DNA polymerase binds to the primer.
- DNA polymerase starts making a new strand of DNA by incorporating free nucleotide bases (A, C, G and T) that are complementary to the DNA on the template strand.
- This continues until two identical copies of the original, double-stranded molecule are produced.
- The DNA double helix is ‘denatured’ (broken down) with heat or chemicals to separate the two strands. These will then act as templates for DNA synthesis.
- A primer, DNA polymerase and nucleotide bases (A, C, G and T) are added. One or more of these bases is radioactively labelled so that any DNA that is synthesised can be detected.
- Other versions of these bases, known as terminators, are also added in small amounts, starting with the ‘A’ terminator. Terminators stop DNA synthesis. So, the ‘A’ terminator will stop DNA synthesis when an ‘A’ base is added (the ‘C’ terminator will stop DNA synthesis when a ‘C’ base is added and so on…)
- This results in a mixture of pieces of radioactive DNA of various lengths but all ending in the same base, in this case, the A base.
- The same reaction is then also set up for the other three bases:
- a mixture of nucleotides plus a C terminator
- a mixture of nucleotides plus a G terminator
- a mixture of nucleotides plus a T terminator.
- The four different reactions are then loaded on to separate lanes of an acrylamide gel and the DNA pieces separated according to size by a process called electrophoresis.
- First they are loaded into separate lanes of a gel (like a slab of firm, transparent jelly).
- Electrodes are placed at either end of the gel and an electrical current is applied.
- DNA is negatively charged, so when the current is applied, the DNA fragments migrate to the positive end of the gel (opposites attract!).
- Small fragments go through the gel quicker than larger fragments
- The radioactively labelled DNA is then visualised by exposing the gel to X-ray film. The radioactively labelled DNA will make the film turn black. This exposed film is called an autoradiogram.
- Each band on the film corresponds to where a specific terminator version of one of the bases has been added (A, C, G or T). You can therefore read off the sequence of the DNA from the bottom of the film.
This page was last updated on 2021-07-21
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