Consider the following DNA sequencing experiment: 3\' ----- GATACCGCTA-5\' (temp

Question

Consider the following DNA sequencing experiment: 3' ----- GATACCGCTA-5' (template)
5' primer

In this set up, DNA polymerase will place a C just after the primer and across from the G in the template strand, and additional bases will be added in the usual manner. Four separate reactions will be run, and each will contain all four dNTPs; however, the "A" experiment will contain, in addition to the four dNTPs, a small amount of ddATP; the "G' experiment will contain all four dNTPs and a small amount of ddGTP, and so on. Given this information, when a sample from the "G" experiment is run on a gel, what will be the sequence (5' to 3') of the smallest complementary strand?

Explanation / Answer

ddNTPs (didioxynucleosidetriphosphate) are stops the activity of DNA polymerase hence it stops the synthesis of new base. So when ddGTP is binds to C the reaction is stopped. This mechanism is widely used in sanger sequencing.

Our sequence is 3’-GATACCGCTA-5’

The complementary sequence for this sequence which is synthesized is: 5'- CTATGGCGAT - 3'

By adding ddGTPs in G experiment 4 fragments are generated

5'-CTATddG-3'

5'-CTATGddG-3'

5'-CTATGGCddG-3'

5'-CTATGGCGAT-3' (this fragment is observed because we also have dNTPS)

On the other hand in experiment A having ddATP fragments observed on gel are

5'-CTddA-3'

5'-CTATGGCGddA-3'

5'-CTATGGCGAT-3'

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