What is the 454 method of DNA sequencing?

• The 454 method can sequence fragments of DNA equivalent to up to one billion bases, in a single day (that’s a 1/3 of the human genome).
• It is much cheaper and faster than the methods used to sequence DNA in the Human Genome Project.
• However, the platform is not without its weaknesses. It has difficulty distinguishing the number of bases in a run of identical bases (such as AAAA).

A 454 sequencing machine.
Image credit: Genome Research Limited

How does 454 DNA sequencing work?

1. The process begins with a sample of double-stranded DNA.
2. The DNA is broken up into fragments of around 400 to 600 base pairs using restriction enzymes that ‘cut’ the DNA at specific points.
3. Short sequences of DNA called adaptors, are attached to the DNA fragments.
4. Tiny resin beads are added to the mix.
5. DNA sequences on the beads are complementary to sequences on the adaptors, allowing the DNA fragments to bind directly to the beads, ideally one fragment to each bead.
6. When the DNA fragments attach to the DNA on the beads the bonds joining the double-strand together, break and the strands separate, becoming single-stranded DNA.
7. The fragments of DNA are then copied numerous times on each bead by a reaction known as the polymerase chain reaction (PCR). This creates millions of identical copies of the DNA sequence.
8. The beads are then filtered to remove any that have either failed to attach to any DNA or contain more than one type of DNA fragment.
9. Then, the remaining beads are put into wells on a sequencing plate (one bead per well) along with enzyme beads that contain the DNA polymerase and primer needed for the sequencing reaction
10. The polymerase enzyme and primer attach to the DNA fragments on the beads.
11. Nucleotide bases are added to the wells in waves of one type of base at a time: a wave of As, followed by a wave of Cs, followed by Gs, followed by Ts.
12. When each base is incorporated into the DNA, light is given out and this is recorded by a camera.
13. The intensity of the light corresponds to the number of nucleotides of the same type that have been incorporated. For example, if there are three consecutive As in the fragment, the amount of light generated would be three times that of a single A in the fragment.
14. By plotting this pattern of light intensity on a graph, the sequence of the original piece of DNA can be decoded.