Question

How does an automated gene sequencer detect the specific nucleotide at the end of a DNA fragment?

Answer 1

# DNA sequencer uses Sanger's dideoxy method. In this method the DNA is chemically modified and the polymerisation will be terminated by fluorescent labeled ddNTPs (dideoxy nucleotide triphosphates). Introduction of these labeled ddNTPs will termiate the DNA polymerisation abruptly. ddNTPS do not have 3'OH group in their ribose sugar. So, they cannot be polymerised further.

From this, the last base will be determined by fluorescent tag. Terminated fragments will be run to separate electrophoretically based on their size. Size of the fragments and the last base will be calculated to predict the actual DNA sequence.