Replication of DNA in Vitro 



327 



been observed that when PP is added to the 

 usual synthesizing complex in great excess 

 (about 100 times the concentration of the 

 triphosphates), the synthetic reaction is in- 

 hibited by about 50%. This suggests the 

 reversibility of DNA synthesis in vitro. 



It was also mentioned earlier that, in a 

 Hmited reaction, £/CP*(PP) could add onto 

 a chain terminating in all four types of 

 nucleotides {dC?, TP, dkV, and dG?). 

 This should not be assumed to mean that 

 dC?* joins linearly to each nucleotide with 

 equal frequency, or that any nucleotide joins 

 to all others with equal frequency. Other 

 results indicate that the limited reaction does 

 not add nucleotides to the end at random; 

 this reaction probably involves the repair of 

 the shorter strand of a double helix, the 

 particular nucleotide added being specified 

 in the usual way by the bases present in the 

 longer strand. 



What is the linear arrangement of nucleo- 

 tides in the DNA synthesized in an extended 

 reaction? We have noted (p. 306) that, if 

 DNA is genetic material, different linear seg- 

 ments of it may represent different genes. If 

 so, the differences among genes would lie 

 in the sequence of the organic bases they 

 contain. Considering only the four usual 

 deoxyribotides, how many different sequences 

 of two nucleotides are possible? The first 

 nucleotide may be one of four, and so may 

 the second, so that there are 4X4, or 16, 

 different possible linear arrangements in di- 

 nucleotides. The orders of dinucleotides can 

 be determined experimentally as follows. 

 One of the four triphosphates added as sub- 

 strate is labeled with P^- in the innermost 

 phosphate, the other three are not. Extended 

 synthesis is permitted, during which the P* 

 attaches to the 3' of the sugar of the nucleo- 

 tide which is its linear neighbor (refer to the 

 left part of Figure 35 3 and Figure 9 in Sup- 

 plement VI). This linear neighbor can be 

 identified by digesting the synthesized prod- 

 uct with micrococcal DNAase and splenic 



phosphodiesterase. You recall that the latter 

 produces deoxyriboside 3'-monophosphates 

 by breaking the chain at 5'. Consequently the 

 labeled phosphate (P*) will be found joined at 

 the 3' position of the deoxyriboside just ante- 

 rior to the one with which it entered the DNA 

 strand. The digest is then analyzed to see 

 how frequently P* is part of dk 3'-P*, 

 T 3'-P*, dC 3'-P*, and dG 3'-P*. If the P* 

 was originally in afAP*PP, this would tell 

 the relative linear frequencies of AA, AT, 

 AC, and AG. If this procedure is carried 

 out three more times, each time labeling a 

 different one of the triphosphates, the relative 

 frequency of all 16 sequences can be deter- 

 mined. 



The results of such studies show that all 

 16 sequences are produced by each type of 

 natural primer, although each primer type 

 has a unique and reproducible pattern, not 

 predictable from its base composition. More- 

 over, if, for example, TG is a common linear 

 sequence, so is CA, while if AG is rare, so 

 is CT. This can be explained by the fact that 

 both strands of a double helix are synthesized 

 in an extended reaction, and that the two 

 chains run in opposite directions. Thus, if 

 TG is the dinucleotide sequence found in one 

 strand, the dinucleotide sequence in the com- 

 plementary strand would be CA. 



All the physical and chemical character- 

 istics of DNA synthesis in an extended reac- 

 tion, including the requirement of all four 

 deoxyriboside 5'-triphosphates and of DNA 

 primer, are consistent with the view that this 

 is a biological process. This means that the 

 synthesis is performed in vitro in essentially 

 the same manner as in the living cell, and 

 produces essentially the same product. It 

 should be emphasized that there are two, 

 perhaps separate, processes involved in the 

 biological replication of DNA. One process 

 results in the side-by-side arrangement of 

 complementary base pairs by means of 

 H-bonding, the other produces the linear at- 

 tachment of nucleotides by the estabUsh- 



