ILLUMINA (SOLEXA) DNA SEQUENCING

Solexa DNA sequencing allows for one base at a time to be identified from a DNA sample via the use of fluorescence.

The DNA sample must firstly be prepared and randomly fragmented to about 100-500bp long. Blunt end ligation is then carried out in order to attach the adaptors-these contain the purification tags for fluorescence. The DNA sample is then washed over a flow cell and the single stranded fragments attach randomly to the inside surface. Nucleotides and enzyme are then added in order to initiate bridge amplification which results in double stranded bridges. Denaturisation must then occur in order to leave single stranded templates.

The first cycle then occurs in which all 4 dNTP's are washed over the flow cell and attach to the first nucleotide base of each DNA strand. These dNTP's have been modified in order to contain a 3'blocking group, which prevents further dNTP's attaching to the adjacent bases. They are also labelled with different fluorescent colours depending on their base. 

After attachment, all free dNTP's can be washed off and a computer image can be taken to identify colours and therefore the first base via complement pairing.

Before the second cycle occurs, the Staudinger reaction and then hydrolysis of hemiaminals occur in order to remove the 3'blocking group and the fluorescent dye. This allows for the next nucleotide base to attach as it is no longer being blocked. Multiple coloured images can be compared from each cycle to identify the sequence of the sample DNA.