454 Pyrosequencing
454 Pyrosequencing involves the chemiluminescent detection of inorganic pyrophosphate release (adenosine triphosphate) and luciferin that occurs on specific nucleotide incorporation into single-stranded PCR products. 454 Life Sciences launched the first next-generation DNA sequencer in the year 2005. This method was originally developed at the Royal Institute of Technology in Stockholm, Sweden in 1996. It was a big leap forward in DNA sequencing technology. The 454 method can sequence fragments of DNA in a single day which is equivalent to one billion bases which are equivalent to 1/3 of the human genome. The array-based method allows sequencing of 400–600 megabases in 10 hours allowing an entire human genome to be sequenced in approximately 27 days. It is much faster and cheaper than the methods used to sequence DNA in the Human Genome Project. Pyrosequencing requires approximately the same time as HRM analysis followed by Sanger sequencing and has high sensitivity, with a limit of detection of ≥ 5% tumor cells within a biopsy sample. But, it has difficulty in distinguishing the number of bases in a run of identical bases (such as AAAA). Additionally, the ability to detect gene copy number changes and structural chromosomal changes is limited.
The 454 pyrosequencing process begins with a sample of double-stranded DNA. The DNA is broken up into fragments of around 400 to 600 base pairs using restriction enzymes that cut the DNA at specific points. Short sequences of DNA called adaptors are attached to the DNA fragments. Tiny resin beads are added to the mix. 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. 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. 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. 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. Then, the remaining beads are put into wells on a sequencing plate along with enzyme beads that contain the DNA polymerase and primer needed for the sequencing reaction. The polymerase enzyme and primer attach to the DNA fragments on the beads. 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. When each base is incorporated into the DNA, light is given out and this is recorded by a camera. 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. By plotting this pattern of light intensity on a graph, the sequence of the original piece of DNA can be decoded.
454 pyrosequencing is a method of high throughput DNA sequencing that utilizes a single strand of DNA with a length of a minimum of 400–500bp. The single strand is used as a template to synthesize the sequence of its complementary strand, further which is determined by a chain of reactions resulting in light being emitted when a specific nucleotide or length of nucleotides are added to the complementary sequence.
