Replication of DNA in Vitro 291 



Sinsheimcr, R. L., •'Single-Stranded DNA," Scient. Amer.. 207 (No. 1): 109-1 16, 1962. 



Spencer, J. H., and Chargaff, E., "Studies on the Nucleotide Arrangement in Deoxyribo- 

 nucleic Acids. VI. Pyrimidine Nucleotide Clusters: Frequency and Distribution 

 in Several Species of the AT-Type," Biochim. Biophys. Acta, 68:18-27, 1963. 



Richardson, C. C. Schildkraut, C. L.. and Romberg, A.. 'Studies on the Replication 

 of DNA by DNA Polymerases," Cold Spring Harb, Sympos. Quant. Biol "»8 9 19 



1 964. 



See Supplement V. A list of references is given at the end of Dr. Romberg's Nobel 

 Prize lecture. 



QUESTIONS FOR DISCUSSION 



21.1. Has this chapter dealt with genetics? Explain. 



21.2. Which single experiment described in this chapter would you consider the most 

 important? Why? 



21.3. Differentiate between the action of splenic phosphodiesterase and snake venom 

 diesterase. 



21.4. What are the requirements for the in vitro synthesis of DNA to proceed as a 

 limited reaction? To proceed extensively? 



21.5. What effect does the absence or presence of pre-existing DNA have upon DNA 

 strand formation in vitro? 



21.6. List the evidence that synthesis of DNA in vitro represents a biological process. 



21.7. Does DNA polymerase from E. coli take directions only from E. coli DNA? 

 Explain. 



21.8. Does strand separation occur during an extended synthesis of DNA in vitro? 

 Explain. 



21.9. Of what significance is the nearest-nucleotide-neighbor analysis? 



21.10. What is wrong with Figure 8 in Supplement V? 



21.11. If substrate depletion is prevented, what would you expect to happen to the 



A + T 



ratio when an extended synthesis is permitted to proceed for several 

 C + o 



hours? 



21.12. How can you explain that an extended synthesis using dGdC as primer-template 

 usually yields a product richer in G than C? 



21.13. List the similarities and differences between the DNA polymerases isolated from 

 E. coli and calf thymus. 



21.14. What is meant by the statement that newly synthesized DNA is covalently linked 

 to the primer but is not covalently linked to the template? 



21.15. State three different procedures which you might use to synthesize dGdC in vitro. 



