4. Consider the amplification of a DNA strand using the indicated single-strand

Question

4. Consider the amplification of a DNA strand using the indicated single-strand DNA (ssDNA), DNA polymerase, excess primer, and a mixture of deoxynucleoside triphosphates (dNTPs) and Mg2+. In this case, normal dATP is replaced in the mixture of dNTPs by 2,3 dideoxyadenosine triphosphate (ddATP). (a) What will be the product? (b) Show the key chemical event that dictates this result. (c) If the ssDNA is that produced by reverse transcription from an RNA virus like HIV (AIDS virus), what would happen by dosing the virus- infected cell with ddA? (10 pts) ssDNA: 3'-ACTGGCCGTCGTTTTACAACGTACGTACGTACGTACGTACGTACGTACGT-5' Primer: 5'-TGACCGGCAGCAAAATGT-3

Explanation / Answer

(a) Polymerase will stop 4 nucleotides from 3’ end at next T

(b) 2',3'-Dideoxyadenosine triphosphate (ddATP) is a sugar modified nucleoside triphosphate, in which 2' and 3' hydroxyl groups are absent, and DNA polymerases cannot extend using incoming ddATP resulting in chain termination. Particularly lack of oxygen at 3’ stops the condensation reaction between 3’ and 5' phosphates. As the 3’ oxygen is missing incoming phosphate on a nucleoside cannot form P-O-P phosphodiester bond.

(c)RNA dependent DNA synthesis requires dATP complementary to Uracil, as there is ddATP DNA cannot be synthesized and virus replication stops.

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