7. You live in the 1970\'s and ran a sequencing analysis using radiolabeled d dN

Question

7. You live in the 1970's and ran a sequencing analysis using radiolabeled d dNTPs. The result of your individual reactions are depicted on the DNA below. (top) (bottom) (a) What is the sequence of your sequenced DNA fragment? Please indicate the 5' and 3' ends, providing an argument as to why you know which end is which (b) At which end of the gel are the smaller fragments of DNA located? Top, or bottom? Provide a brief explanation as to why the fragments separate in this manner (c) Fast forward to today. Let's say that you use the modern fluorescent ddNTP method and are able to determine a 700 base pair sequence of your analyzed DNA. However, 700 base pairs is not enough, and you want to know the sequence of the DNA beyond this this region. A friend suggests that you perform the sequencing reaction under conditions in which the ratio of ddNTP:dNTP is increased. Would this increase the length of your sequencing read? Explain why or why not.

Explanation / Answer

A)5' TAGATCTGACCTG 3'

The replication starts from 5' to 3' and that is why the first base that is the base is 5' and it goes towards 3' of the end.

B) The bottom part is the part where the smaller fragments are located. The gel has certain pore size and from that pore size only smaller fragments can elute first and that is why the small fragments are always located on the bottom.

C) No, it won't increase the length of your sequencing read because the solution has higher amount of ddNTPs and polymerase can not add ddNTPs as it does not have 3'OH.

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