The Human Genome Project

Developing the Deoxy Chain Terminiation Sequence

March 2009

And taking advantage of this is how the deoxy chain termination sequencing was developed. If one takes a very low concentration of a dead end inhibitor, a dideoxy A, and adds it to the reaction, perhaps 1:1000 is the ratio between the dideoxy-A and the normal deoxy-ATP, so that most of the time the base is added and that base enables an additional base to be added. But when the dideoxy base is added, that stops the chain from growing. If one has 4 separate reactions (a dideoxy A, a dideoxy G, a dideoxy C, and a dideoxy T) those separate reactions can be used to monitor the growing of the chain. What happens is that sometimes you will stop at the first available A, sometimes you will stop at the second, at the third, etc. and what happens is you end up generating a ladder. These bases were originally put in and they were radioactive so the growing chains were radioactive and the growing chains could be detected with autoradiography. So after the gel is run and a piece of radiographic film is superimposed on top of this and then developed, you can actually see the bands where the chain has terminated at the very first A, the next available G, and then you see 2 where C’s have been added. One literally reads up this ladder to determine the sequence; you can get a sequence of the growing chain from this and from that sequence you can infer the sequence of the original molecule that you started with.

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