454 Sequencing is a type of massively parallel DNA sequencing. 454 sequencing uses a large-scale system known as PYROSEQUENCING and able to sequence much longer reads than Illumina sequencing. 454 Sequencing was created by biotech company 454 in 2005 and was released as a faster, cheaper alternative to other DNA sequencing methods available at the time, such as the Sanger “dideoxy” method.
Steps of 454 Sequencing:
- Random fragmentation of DNA.
- Ligation of common adaptor sequences to each end of the fragmentized DNA.
- The double stranded DNA is denatured to form single stranded DNA by using NaOH.
- The single strands mixed with the capture beads which contain DNA sequence that complementary to one of the adaptor, allowing the DNA fragments to bind directly to the beads.(Takes note that only one fragment of DNA can be caught per bead).
- Once the DNA fragment attach to the bead, it starts adding the nucleotide sequence which is complementary to the DNA fragement.
- Then, the hydrogen bonds break, causing the double strands to seperate, forming single-stranded DNA.
- The fragments of DNA are then copied numerous times on each bead by polymerase chain reaction (PCR).This helps to create millions of identical copies of the DNA sequence.
- The beads are put into the wells of the picotitre plates with DNA immobilised on beads.
- The polymerase enzyme and primer attach to the DNA fragments on the beads.
- Nucleotide bases such as dATP, dGTP, dTTP and dCTP are sequencially added into the picrotitre plate.
- A light signal is generated once each base is incorporated into the DNA.
- These light signals is detected by a detector and captured by a camera.
- ↑ Wellcome.ac.uk, (2015). DNA sequencing - the 454 method | Wellcome Trust. [online] Available at: http://www.wellcome.ac.uk/Education-resources/Education-and-learning/Resources/Animation/WTX056046.htm [Accessed 19 Oct. 2015].
- ↑ Berg J, Tymoczko J, Stryer L. Student companion for Biochemistry 7th edition, international edition:Protein synthesis.7th ed. New York: W.H. Freeman; 2011.
- ↑ Yourgenome.What is the 454 method of DNA sequencing.2015[cited 25/2/15]Available from:https://www.yourgenome.org/facts/what-is-the-454-method-of-dna-sequencing