Question

P-P-P-O-CH2 deoxycytidine triphosphate (dCTP) OH H P-P-P-O-CH2 dideoxycytidine triphosphate (ddCTP) NH2 P -0-CH dideoxycytidine monophosphate (ddCMP) HH A. What would you expect if ddCTP were added to a DNA replication reaction in large excess over the concentration of the available CTP, the normal deoxycytidine triphosphate? B. What would happen if it were added at 10% of the concentration of the available dCTP? C. What effects would you expect if ddCMP were added under the same conditions?

Answer 1

A. The dideoxycytidine triphosphate (ddCTP) is similar to deoxycytidine triphosphate (dCTP). However, it lacks the 3’ hydroxyl group that is present in the deoxyribose sugar in dCTP. DNA polymerase requires this 3’ OH group to incorporate the nucleotide into growing DNA strand. If DNA polymerase encounters ddCTP, instead of dCTP, it will still recognize ddCTP as dCTP and tries to incorporate into growing DNA strand. However, as there is no 3’ OH group, it cannot add ddCTP to the growing strand resulting in chain termination. Hence, whenever the DNA polymerase encounters the first guanine in the template strand, it will try to incorporate ddCTP instead of dCTP into the growing strand. This results in premature termination of DNA replication, resulting in no DNA synthesis occurring right from the start. As a result, no new DNA strands/ fragments will be produced.

B. The ddCTP is added at a 10% concentration of dCTP. Hence, there is a 1 in 10 chance that DNA polymerase will use ddCTP instead of dCTP for DNA replication when a guanine is encountered on template strand. This results in formation of DNA fragments of different sizes. Thus, a number of DNA fragments of different length based on the position/ location at which guanine was encountered by DNA polymerase will be synthesized. The length of these fragments can therefore help to determine where G (or C in growing strand) is present on DNA sequence in template. This phenomenon is the basis of Sanger Sequencing (and other sequencing techniques) that is used to detect the sequence of nucleotides in DNA.

C. The ddCMP is dideoxycytidine monophosphate. In addition to lack of the 3’ OH group on deoxyribose sugar, it also lacks two phosphate groups. Hydrolysis of phosphate groups in dNTPs provides the free energy for addition/polymerization of nucleotides by DNA polymerase during DNA replication (forming the phosphate sugar backbone). Due to lack of phosphate groups, no energy will be available for polymerization during replication. Thus, ddCMP or even dCMP cannot act as a substrate for DNA polymerase. Hence, it should not affect DNA replication at excess or 10% concentration.