A) What is the difference between dNTPs and ddNTPs? (1 sentence answer) B) What

Question

A) What is the difference between dNTPs and ddNTPs? (1 sentence answer)

B) What function do dideoxynucleotides (ddNTPs) have in Sanger DNA sequencing? (1 sentence answer)

C) Write the entire sequence of the product molecule corresponding to the band indicated by the arrow (and indicate 5’ and 3’ ends). Underline any incorporated base that is fluorescently labeled.

Part II. DNA sequencing (2.5 points) You now wish to sequence DNA from the patient's CEBPA gene and first do a control experiment to make sure your sequencing reaction works (you will need to consult Griffiths p. 374-377, for an explanation of DNA sequencing and dNTPs versus ddNTPs, also look at: https://www.youtube.com/watch?time_continue-89&v-e2G5zx-OJlw;). You set up a DNA- sequencing reaction with the following primer and template and include dNTPs as well as a small amount of fluorescently labeled ddNTPs (ddATP-red, ddTTP-green, ddCTP-blue, ddGTP. orange) Primer: 5'-GCCTCGTCAT-3' Template: 3'-CGGAGCAGTANNNNNNNNNNNNNNNN-5' (where N is an unknown base) 10 uM dATP, dTTP, dCTP, dGTP 1 uM ddATP, ddTTP, ddT dTP DNA polymerase You place the reaction products into a capillary tube electrophoresis chamber and apply a voltage. DNA moves toward the positive electrode through the medium such that smaller DNA fragments move faster than larger DNA fragments. The result is that DNA becomes separated by size, with large DNA fragments at the top of the gel and small DNA fragments at the bottom of the gel. For analysis of the sequencing reaction, the DNA is denatured prior to separation

Explanation / Answer

A. dNTPs does not have OH group at 2' sugar, where as ddNTPs dones not have OH group at 2nd and 3 rd positions of the sugar, hence the ddNTPs reffered as 2',3' dideoxyribonucleotides.

B. In DNA, for chain elongation, the 3'OH group is required. As due to absence of this OH group at 3' carbon of the sugar, they will terminate the elongation reaction.

c. Find the below image.

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