Question

In gel electrophoresis why are two different types of nucleotides (deoxyribonucleotides and dideoxyribonucleotides) to the sequencing reaction? Why are the dideoxynucleotides the only ones fluorescently labeled?

Answer 1

In DNA sequencing, chain termination (discovered by sanger), deoxy nucleotide is use to amplify the DNA sequence where as so that a long read of DNA can be read. In deoxy nucleotide 3’-OH group is present (see the below mentioned figure) that synthesize the DNA. On the contrary dideoxy doesnot contain any 3’-OH group in sugar (see the figure) hence this will not propagate the PCR reaction. Hence both the nucleotides are present in the DNA sequencing mixture. But once the PCR sequencing is terminated than how to detect at which base the chain termination sequencing is terminated. Hence these only di-deoxy bases are fluorescently labeled so that they can be detected in capillary electrophoresis.