454 pyrosequencing

454 sequencing is a new technique that allows the researcher to get hundreds of thousands of sequences in a single experiment. The main drawbacks of this method are that (1) the sequences obtained are short, less than 300 bp, and (2) it's expensive (per run, not per sequence) and requires one-of-a-kind machines.

454 sequencing starts with a standard PCR using primers that will generate a relatively short (<300bp) product. Each of the primers have extra sequences on the end, one for annealing to beads, the other serves as a primer binding-site for the sequnecing reactions.

The DNA is denatured, and mixed with a numerical excess of small beads coated with oligonucleotides that bind to one end of the PCR product. Because there is an excess of beads, most of the beads get only one (or none) DNA molecule annealed to them. The slurry of beads in PCR reaction mixture is then emulsified with oil. Each bead ends up in a droplet of PCR reaction solution in this oil emulsion - each of these droplets is a microscopic PCR reaction tube. The emulsion is run through a set of PCR reaction cycles as usual. The PCR product generated in each drop annals to the tags on the bead, so each bead ends up covered in DNA with a single sequence. The emulsion can then be broken (separating the oil and aqueous phases), an the DNA-covered beads separated out.

The beads are then washed over a silicon wafer honeycomb "picotiter plate". Each cell of the honeycomb is big enough for one and only one bead, so the honeycomb ends up filled with one bad per "well". Each of these then serve as a sequencing reactor. The wells are filled with very small beads coated in 2 enzymes: sulfurylase and luciferase.

The honeycombs are then percolated with reaction mixture of the sequencing primer, DNA polymerase, and APS (adenosine 5'-phosphorothioate) and luciferin, followed by sequential rounds of dGTP, dATP, dCTP and dTTP, over and over again. When the next nucleotide in the sequence matches the dNTP added, the base is added to the growing DNA chain, releasing pyrophosphate. Sulfurylase replaces the thio grup on APS with the pyrophosphate, creating ATP, which is used by luciferase to react with luciferin to generate a pulse of light.

In other words, the well "blinks" a flash of light to signal that the next nucleotide in the sequence matches the current dNTP. If there is a stretch of 2 or more of the same base, proportionally more pyrophosphate is generated, and the flash of light is proportionally stronger. So, as the nucleotides flow past in repeated sequence, each well signals the sequence of the DNA on that bead in a kind of Morse code. A magnifying CCD video camera watches the honycomb, collecting the data from all of the wells over time, and a computer then separates the data from each spot to read the sequences.

(this image is false-colored red, green blue and yellow for each base in a single round of sequencing)

BTW, "454" apparently comes from the address of the startup company that developed this method, although others have suggested that it comes from the the 'fact' that this is the temperature at which money burns. Refinement of the techonology is increasing the length of the sequence reads (last year I gave the maximum sequence length s 100), and there is no conceptual reason why they couldn't ultimately be as long on average as regular Sanger sequencing methods.