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

Question

7. You live in the 1970's and ran a sequencing analysis using radiolabeled ddNTPs. The result of your individual reactions are depicted on the DNA below. (top) 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 (a) 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. (b) (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) Sequence of the DNA is 5'TAGACTGACCTG3'.

In this technique, ddNTP's are used. These ddNTP's lack the 3'-OH goup that is essential for the elongation of the DNA strand during PCR reactions. Hence as soon as such an ddNTP is encountered, elongation of the strand in the PCR is stopped.

we know that during PCR elongation, dNTP's are added from the 5' end. Now during a PCR reaction, if at a very early elongation stage, a ddNTP is encountered, a small ampliefied DNA is obtained. On a separating gel, this DNA fragmnet is always found at the bottom of the gel. Hence, seqeunce of the DNA at the 5' are found at the bottom of the separating gel.

Similarly, longer fragments are found towards the top of the gel and hence these dNTP's lie at the 3' end of the DNA sequence.

B) smaller fragments are always found at the bottom of the gel, where as the longer fragments are closer to the top. This is due to hte fact that smaller fragments can wriggle easily through the pores of the gel and encounter less friction. Hence they can easily move though the gel.

But for longer fragments , due to the increase in size encounter greater friction (as they cant pass through the pores of the gel as easily as the smaller fragments) and hence get stuck near the top of the gel.

C) Yes by increasing the ratio of ddNTP:dNTP, the length of the sequencing read. As more dNTPs are available for the enzyme to add during the elongation step, longer fragments can be obtained. However, the sequence obtained by thsi technique will not be that accurate.

A better approach would be to start another seuencing run from the point till which the analysis was done previously.

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