You are studying DNA polymerase ? (pol ?) from the thermophile, B. usfveryhotili

Question

You are studying DNA polymerase ? (pol ?) from the thermophile, B. usfveryhotilis. Pol ? is very similar to pol III from E. coli, except that activity of pol ? is exactly the same from 10oC to 98oC, a very broad temperature range! You mix pol ? with dATP, dTTP, dGTP, dCTP, Mg(II), and a DNA fragment with the sequence, 5’-TTTCACAGGTACTTTTGTACC-3’, and allow the reaction to incubate for 1 hr at 15oC.

What is the product of this reaction?

Explain why these are the answers. 1 is D and 2 is A

I. You are studying DNA polymerase ? (pol ?) from the thermophile, B. usfveryhotilis. Pol ? is very similar to pol III from E. coli, except that activity of pol ? is exactly the same from 10°C to 98°C, a very broad temperature range! You mix pol ? with dATP, dTTP, dGTP, dCTP, Mg(II), and a DNA fragment with the sequence, 5'-TTTCACAGGTACTTTTGTACC-3', and allow the reaction to incubate for 1 hr at 15°C. What is the product of this reaction? Assume that the concentration of dATP, dTTP, dGTP, and dCTP are not limiting. A. No reaction, because no primer was included. B. 5'-AAAGTGTCCATGAAAACATGG-3' C.5'-GGTACAAAAGTACCTGTGAAA-3' D. 5'- TTTCACAGGTACTTTTGTACCTGTGAAA-3' E. 5'- TGTGAAA-3' 2. You are studying DNA polymerase ? (pol ?) from the thermophile, B. usfveryhotilis. Pol ? is very similar to pol III from E. coli, except that activity of poly is exactly the same from 10°C to 98°C, a very broad temperature range! You mix pol ? with dATP, dTTP, dGTP, dCTP, Mg(11), and a DNA fragment with the sequence, 5'-TTTCACAGGTACTTTTGTACC-3', and allow the reaction to incubate for 1 hr at 95°C. What is the product of this reaction? Assume that the concentration of dATP, dTTP, dGTP, and dCTP are not limiting. A. No reaction, because no primer was included. B.5'-AAAGTGTCCATGAAAACATGG-3' C.5'-GGTACAAAAGTACCTGTGAAA-3' D.5'- TTTCACAGGTACTTTTGTACCTGTGAAA-3" E. 5'- TGTGAAA-3'

Explanation / Answer

1) D

AT low temperature deoxyribonucleotide in the form of dATP, dGTP, dCTP and dTTP are randomly able to attach at the 3' end of DNA. SO it will be randomly added at the 3' end of existing DNA in vitro because of free OH group at 3rd carbon of ribose sugar. This is not the replication.

2(A) At high temperature simply addition of nucletide is not possible. High temperature promote depolymerisation. If DNA strand is single stranded or deploymerise at high temperature and it want to go for replication process then it need primer for its synthesis. Since experiment is carried out in Vitro (outside cell), then without primer, synthesis of complememtary strand is not possible. Hence no reaction occurs.

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