Replication of DNA in Vitro 



281 



P-P--P 



* 



>DNA 



>DNA 



figure 21-2. Growth of a primer DNA strand at its nucleoside end (left) and nucleo- 

 tide end (right). Arrows show 5' positions of subsequent degradation by micrococcal 

 DNase plus splenic phosphodiesterase. 



DNA may come from a plant, animal, bac- 

 terium, or virus. Similar DNA-synthesizing 

 extracts can be prepared from other bacteria, 

 calf thymus, and various animal tissues. 



Extended and Limited Syntheses 



Using E. coli preparations, we can obtain 

 an extended synthesis of DNA which pro- 

 duces twenty or more times as much DNA 

 as was initially present. In this case, there- 

 fore, 95% or more of the DNA present at 

 the end must have been synthesized from 

 the triphosphates added as substrate, the ex- 

 tended synthetic reaction proceeding until 

 the supply of one of the four triphosphates 

 is exhausted. One inorganic pyrophosphate 

 is released for each deoxyribotide incorpo- 

 rated into DNA. 



Although extensive synthesis of DNA does 

 not occur if only one of the deoxyriboside 

 5'-triphosphates is added as substrate, some 

 incorporation of this nucleotide into the 



DNA strand occurs in what is called a lim- 

 ited reaction. By what mechanism does the 

 nucleotide add on to the pre-existing DNA 

 strand? In this case, the already-present 

 DNA must provide a point of linear attach- 

 ment for newly-forming DNA, thereby func- 

 tioning as a primer. Suppose that the only 

 triphosphate added to the substrate is de- 

 oxycytidine 5'-triphosphate whose innermost 

 phosphate carries radioactive P A - (dCP*PP). 

 The two possible ways in which the DNA 

 strand might lengthen are shown at the left 

 and right of Figure 21-2; the DNA strand, 

 present as primer, is shown enclosed in 

 brackets. The primer strand can be con- 

 sidered to have a nucleotide end (top of the 

 figure) — to which pyrophosphate, P-P, is 

 added in the diagram to the right — and a 

 free 3'-OH nucleoside end (bottom of illus- 

 tration). (The removal of a sugar and base 

 by a single break at the 5' position involves 

 the removal of a nucleoside at the nucleoside 



