Replication of DNA in Vitro 



321 



any synthetic process which may be taking 

 place. The problem of identifying DNA syn- 

 thesis in the absence of a net increase in DNA 

 quantity can be solved by preparing the 

 deoxyriboside thymidine, which has radio- 

 active C'^ incorporated in its pyrimidine, and 

 adding this substrate to the extract. If any 

 radioactively labeled thymidine is incorpo- 

 rated into the DNA of the extract, it probably 

 would do so by a synthetic reaction, since, 

 you recall, there is little incorporation into 

 DNA except during synthesis. 



Finally, we ought to obtain our extract 

 from cells that are growing and dividing 

 rapidly, so that they are hkely to contain a 

 maximum amount of apparatus for DNA 

 synthesis, which is being used at full capacity. 

 In line with this reasoning and after such 

 preparation,^ an experiment was performed 



' The preceding and following account is based pri- 

 marily upon work by A. Kornberg and his associates. 



with an extract of the bacterium Escherichia 

 coli. After a suitable time interval (about 30 

 minutes) during which extract, radioactive 

 thymidine, ATP, and Mg ions were incubated 

 at an appropriate pH, the pH was then made 

 suitably acidic, under which condition DNA 

 is precipitated but deoxyribonucleosides {de- 

 oxyribosides) remain soluble. The acid pre- 

 cipitate was washed many times until it was 

 certain that the DNA precipitate was not 

 contaminated by adsorbed deoxyribosides. 

 The precipitate was then examined and found 

 to be only slightly radioactive (50 counts as 

 compared with the 5 million counts present 

 in the added thymidine substrate)! In fact, 

 the amount of thymidine incorporated was so 

 small that it would have been 10,000 times 

 too small to be detected by ordinary chemical 

 analysis. Nevertheless, this radioactivity was 

 doubtless due to thymidine which had been 

 incorporated into the DNA, as could be 



A R P P P 



N R P ~ 



ADENOSINE NICOTINAMIDE 



TRIPHOSPHATE MONONUCLEOTIDE 

 (ATP) 



ARPPRN + PP 



DIPHOSPHOPYRIDINE 

 NUCLEOTIDE 



A R P P P 



F R P 



FLAVIN 

 MONONUCLEOTIDE 



: ARPPRF + PP 



FLAVIN ADENINE 

 DINUCLEOTIDE 



HjR 



+ P P 



