The provided images are the problem and the actual answer key. What I want to kn
ID: 181806 • Letter: T
Question
The provided images are the problem and the actual answer key. What I want to know is how did they get the answer? what was the process and what are the steps to achieve the bands on the gel?
PROBLEM
ANSWER
11. (4 points) The DNA shown below is to be sequenced. In the gel diagram, draw in the bands that will appear if DNA polymerase is added to the reaction along with the nucleotide mixtures indicated. Dideoxynucleotides (ddNTPs) are added in relatively small amounts. The asterisk represents a radioactive label. *5 --3'OH 3 ----ACGACGCAAGGACATTAGAC Nucleotide mixtures: A. dATP, dTTP, dCTP, dGTP, ddTTP (given)Explanation / Answer
ddNTPs can block the synthesis because it lacks OH at 3' position so the normal nucleotide cannot be added when ddNTP get inserted instead of normal nucleotide.
Complementary sequence for the given sequence is synthesized by adding DNA polymerase, dNTPs and ddTTPs in the mixture A.
Given sequence : ACGACGCAAGGACATTAGAC
Complementary sequence: TGCTGCGTTCCTGTAATCTG
Mixture A: contains all dNTPs along with ddTTPs. So ddTTPs can block the T at 8 positions giving rise to 9 bands that is with one complete sequence.
1st band : TGCTGCGTTCCTGTAATCTG
2nd band : TGCTGCGTTCCTGTAATC ddTTP
3rd : TGCTGCGTTCCTGTAA ddTTP
4th : TGCTGCGTTCCTG ddTTP
5th : TGCTGCGTTCC ddTTP
6th : TGCTGCGT ddTTP
7th : TGCTGCG ddTTP
8th : TGC ddTTP
9th : ddTTP
Mixture B: Contains only dATP, dTTP, dGTP and ddCTP. There is no normal CTP so the synthesis get stopped with the very small sequence TG ddCTP. As this is relatively small in size, this can reach the end of the gel and thats why the band is indicated at the end.
Mixture C : Contains dCTP, dTTP, dGTP, ddCTP and ddATP but it lacks normal ATP.
Normal complementary sequence: TGCTGCGTTCCTGTAATCTG
1st band : TGCTGCGTTCCTGTddATP
2nd band : TGCTGCGTTCddCTP
3rd band : TGCTGCGTTddCTP
4th : TGCTGddCTP
5th : TGddCTP
Fragments or DNA move according to the size, smaller the size it can reach the end and greater the size it remains at the top.
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