A DNA fragment was sequenced using dye-terminator sequencing. In this procedure,

Question

A DNA fragment was sequenced using dye-terminator sequencing. In this procedure, ddATP was tagged with a green dye, ddCTP was tagged with a blue dye, ddGTP was tagged with a yellow dye, and ddTTP was tagged with a red dye. The primer used had the sequence 5'-ATGAC-3'. Given the result shown below, what was the sequence of the DNA fragment (template strand)? Note that the shortest fragments are at the bottom and the longest fragments are at the top.

I just don't want the answer. I want someone to explain this to me, I don't even know where to start this problem.

Explanation / Answer

The synthesized strand will be 5'-ATGACGGCCTTTTCTATCGC-3'.

The template strand for the synthesized DNA strand will be 3-TACTGCCGGAAAAGATAGCG-5'.

Dideoxy-dye terminator sequencing method involves the use of ddNTPs which were incorporated into DNA strand being synthesized. When ddNTPs encounter the DNA strand, the chain is terminated since they are not able to form phosphodiester bond with the next dNTP. ddNTPs are used in 1% of total dNTP concentration which allows to form various length of DNA strands. These DNA strands are then separated on agarose gel electrophoresis. The shorter the fragment is longer it moves in the gel. Therefore the autoradiogram explains from bottom to top with polarity of 5'->3' for the newly synthesised DNA strand.

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