Clones; Transformation; Strand Recombination in Vitro 



301 



Strand Recombination in Vitro 



Heating chromosomal DNA denatures it by 

 causing strand separation. After quick cool- 

 ing, the resultant single strands of denatured 

 DNA can fold forming a considerable num- 

 ber of complementary base pairs between 

 bases at different levels of the single strand. 

 It should be noted that, under certain con- 

 ditions, homopolymers of RNA containing 

 A, U, C, or inosinic acid are capable of base- 

 pairing after folding, thus forming regular 

 double-helical structures. To understand 

 de novo synthesis and limited reaction in 

 vitro it may be important to learn the ex- 

 tent to which intrastrand base-pairing oc- 

 curs in DNA homopolymers containing C 

 or G. After pneumococcal DNA is heated 

 for ten minutes at 100° C, all strands are 

 essentially single and all H bonds, broken; 8 

 this denaturation, called melting, occurs 

 sharply at 71° C for dAT and at 83° C for 

 dGdC. When DNA denatured by heat is 

 cooled slowly, only about 70% renaturation 

 occurs for native DNA, although, as ex- 

 pected, dAT and dGdC apparently show 

 100% renaturation. 



Renatured and native DNA differ from 

 denatured DNA in several properties: 



1. Under the electron microscope, rena- 

 tured DNA looks very much like na- 

 tive DNA, whereas denatured DNA is 

 irregularly coiled with clustered re- 

 gions 



2. Renatured and native DNA have sim- 

 ilar and lighter densities than dena- 

 tured DNA 



3. Renatured and native DNA have about 

 twice the molecular weight of dena- 

 tured DNA 



4. Although all DNA has the same ab- 

 sorption spectrum, renatured and na- 



8 The following account is based primarily on work 

 reported by P. Doty, J. Marmur, J. Eigner, and 

 G. Schildkraut (1960), and J. Marmur and D. 

 Lane (1960). 



tive DNA absorb less ultraviolet than 

 denatured DNA. 



Several factors affect renaturation: 



1 . The concentration of DNA in a slowly 

 cooling mixture. When the concentration 

 of single strands is high, so is the amount of 

 renaturation; when the concentration is low, 

 slow cooling does not produce any substan- 

 tial recombination of strands. 



2. Salt concentration. The negatively- 

 charged phosphate groups of single strands 

 tend to prevent union with other strands. 

 This inhibition can be overcome by adding 

 KC1 to the solution to act as a shield against 

 the repulsion between phosphates. Conse- 

 quently, within a certain range, the more 

 KC1 present, the greater the amount of re- 

 naturation obtained by slow cooling of 

 heated DNA. 



3. The source of DNA. Assuming the 

 molecular weight of native DNA to be ap- 

 proximately the same in all organisms, a 

 mammalian cell, which has about a thousand 

 times as much nuclear DNA as a bacterial 

 cell, also has about a thousand times as many 

 DNA molecules. Assuming that all the 

 DNA molecules within a genome differ in 

 base sequence, then, in a given concentra- 

 tion of denatured DNA, on the order of one 

 thousand times fewer complementary strands 

 are present in a sample from calf thymus 

 than in one from Pneumococcus. When 

 equal concentrations of denatured DNA are 

 heated to 80° C, double strands are formed 

 by a large fraction of the bacterial DNA, 

 but by no detectable fraction of the calf 

 thymus DNA. The concentration of com- 

 plementary strands, therefore, is important 

 in renaturation. 



DNA can be denatured in vitro by a large 

 number of organic chemical substances in- 

 cluding urea, aromatic compounds, and a 

 variety of alcohols. This finding, however, 

 does not necessarily mean that such com- 

 pounds have this function in vivo, or that 



