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Illumina dye sequencing is a technique used to determine the series of base pairs in
DNA. It was developed by
Solexa and is based on reversible dye-terminators that enable the identification of single bases as they are introduced into growing DNA strands. It is often employed to sequence difficult regions, such as homopolymers and repetitive sequences. As well, it can be used for whole-
genome and region sequencing, transcriptome analysis, small
RNA discovery,
methylation profiling, and genome-wide
protein-
nucleic acid interaction analysis.[1][2]
Procedure
DNA molecules are attached to
primers on a slide and amplified so that local colonies are formed.
The four types (A, C, T, G) of reversible terminate bases are added, each fluorescently labeled with a different color and attached with a blocking group.
The four bases compete for the binding site and non-incorporated molecules are washed away.
After each synthesis a laser is applied resulting in the removal of the 3’ terminal blocking group and the probe generating a detectable color that is associated with each base allowing for the next cycle.
This technique offers a number of advantages over traditional sequencing methods. For instance, due to the automated nature of illumina dye sequencing it is possible to sequence multiple strands at once and gain actual sequencing data quickly. Whereas,
Sanger sequencing can only characterize one template at a time and sequences have to be separated using
polyacrylamide-
urea gel, which is time consuming. Additionally, it only uses DNA polymerase as opposed to multiple, expensive
enzymes required for other sequencing techniques (i.e.
Pyrosequencing).[3]