Which end of the gel (top or bottom) corresponds to the 5 end of the product (no

Question

Which end of the gel (top or bottom) corresponds to the 5 end of the product (not the template) of the sequencing reaction; that is, the sequence closest to the primer? Why? Determine the nucleotide sequence of the template DNA from the banding pattern in the autoradiogram to the right Determine the sequence of only the region contained in the square. Write your answer in the space below in the 5 to 3 orientation. In the rectangle below, draw the autoradiograph derived from sequencing of the following template strand sequence - ATGGCTAAGATCAGG

Explanation / Answer

1. Top corresponds to the 5' end. The sequencing methods use 5' end as a base. We either label it with 32P or use it as a site for primer binding site.

Hence cleavage by chemical or enzyme will start from 3' end and the lower molecular weight bases moves faster than the others.

In the gel, the sequence is read from bottom to top which corresponds to the 5' to 3' sequence of the template. The complementary sequence will give the exact protein sequence which runs from 3' to 5' from bottom to top. Hence top will give the details of 5' end.

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