Molecular Biologist's Guide
Molecular Biology 344 fall 2012
A Johnson
Due 9/11/12 as hard copy at the beginning of lecture
Problem Set 1
1. Look carefully at the structures of the two molecules shown below. a. What would you expect to happen if you added ddCTP (shown in a) to a DNA synthesis reaction in vitro in large excess over the concentration of deoxycytidine triphosphate (dCTP)? How will this affect the pattern of bands on the sequencing gel? The ddCTP lacks the OH, which is required for elongation of the DNA strand. As a result, when ddCTP is added, elongation will be halted. The ddCTP acts as a chain terminator. The pattern of the bands will be much shorter(as soon as the ddCTP is added, there will be an termination of bands) b. What would happen if ddCTP were added at 10% the concentration of the dCTP? If ddCTP, the chain terminator is added at 10% the concentration of the normal dCTP, there may be a decreased level of polymerization. However, there is little chance of elongation becoming halted, due to the lower concentration of ddCTP. Also, even if the elongation is halted, polymerization could begin again when the ddCTP is removed. c. What effect would you expect if ddCMP (shown in b) were added to the synthesis reaction in large excess over dCTP? At 10% the concentration of dCTP? Nothing will occur. As ddCMP acts as the chain terminator, the probability of getting ddCMP inside is fairly high due to the higher concentration. At 10% the concentration of dCTP, there will be an even smaller (very little) decrease in polymerization.
2. Cystic fibrosis is an inherited chronic disease that affects the lungs and digestive system of about 30,000 children and adults in the United States (70,000 worldwide). A defective gene and its protein product cause the body to produce unusually thick, sticky mucus that: • clogs the lungs and leads to life-threatening lung infections; and • obstructs the pancreas and stops natural enzymes from helping
A Johnson
Due 9/11/12 as hard copy at the beginning of lecture
Problem Set 1
1. Look carefully at the structures of the two molecules shown below. a. What would you expect to happen if you added ddCTP (shown in a) to a DNA synthesis reaction in vitro in large excess over the concentration of deoxycytidine triphosphate (dCTP)? How will this affect the pattern of bands on the sequencing gel? The ddCTP lacks the OH, which is required for elongation of the DNA strand. As a result, when ddCTP is added, elongation will be halted. The ddCTP acts as a chain terminator. The pattern of the bands will be much shorter(as soon as the ddCTP is added, there will be an termination of bands) b. What would happen if ddCTP were added at 10% the concentration of the dCTP? If ddCTP, the chain terminator is added at 10% the concentration of the normal dCTP, there may be a decreased level of polymerization. However, there is little chance of elongation becoming halted, due to the lower concentration of ddCTP. Also, even if the elongation is halted, polymerization could begin again when the ddCTP is removed. c. What effect would you expect if ddCMP (shown in b) were added to the synthesis reaction in large excess over dCTP? At 10% the concentration of dCTP? Nothing will occur. As ddCMP acts as the chain terminator, the probability of getting ddCMP inside is fairly high due to the higher concentration. At 10% the concentration of dCTP, there will be an even smaller (very little) decrease in polymerization.
2. Cystic fibrosis is an inherited chronic disease that affects the lungs and digestive system of about 30,000 children and adults in the United States (70,000 worldwide). A defective gene and its protein product cause the body to produce unusually thick, sticky mucus that: • clogs the lungs and leads to life-threatening lung infections; and • obstructs the pancreas and stops natural enzymes from helping