Replication of DNA in Vitro 



283 



age is the same when net DNA is greatly 

 increased as it is when the limited reaction 

 occurs. 



Characteristics of Synthesized DNA 



That a DNA primer strand can be length- 

 ened in vitro, no matter which of the four 

 common deoxyribosides happens to be at 

 the nucleoside terminus, is consistent with 

 the independence of DNA primary structure 

 upon base sequence. Consider the evidence 

 that the DNA synthesized in vitro has the 

 characteristics of DNA synthesized in vivo. 

 The physical characteristics of DNA samples 

 consisting of 90% or more of the product 

 synthesized in vitro are similar to those of 

 DNA isolated from calf thymus insofar as 

 sedimentation rate and viscosity are con- 

 cerned. Such results indicate that the prod- 

 uct has a high molecular weight (about six 

 million) and, usually, is not single-stranded. 

 In support of the latter inference is the find- 

 ing that the macromolecular structure of the 

 in vitro product is destroyed when heated 

 for 10 minutes at 100° C, an expected result 

 if this treatment is to produce single strands 

 — denatured DNA which collapses to form 

 compact, randomly-coiled structures. Like 

 thymus DNA, the enzymatic product shows 

 the same type of increase in ultraviolet ab- 

 sorption following digestion with pancreatic 

 DNase. 



If the synthesis in vitro occurs as it does 

 in vivo, we might expect single-stranded 

 DNA to serve in in vitro synthesis as well 

 as, or better than, double-stranded DNA. 

 In fact, the single-stranded DNA, isolated 

 from the virus </>X174, is excellent for this 

 purpose, and heat-treated DNA is better 

 than unheated DNA. Moreover, the prep- 

 arations containing the most active DNA 

 polymerase do not work well with double- 

 stranded DNA unless it is first heated or 

 treated with DNase. The DNA produced 

 in extended syntheses behaves as though it 

 is primarily two-stranded, but differs from 



native double-stranded DNA by appearing 

 to be markedly branched in electron micro- 

 graphs and by being readily renatured after 

 heat or alkaline denaturation. These results 

 suggest that strand separation is usually in- 

 complete in the in vitro system. 



In view of these results, we can conclude 

 that the physical characteristics of the DNA 

 synthesized in vitro and in vivo are extremely 

 similar, though not identical. Synthesis 

 clearly involves single strands which pro- 

 duce double strands probably held together 

 by H bonds. 



One can also study the detailed chemical 

 and physico-chemical characteristics of the 

 in vitro synthesis of DNA. If single strands 

 produce double strands by forming comple- 

 mentary structures, then the capacity to form 

 a complementary strand should depend upon 

 the presence in the substrate of purine and 

 pyrimidine bases which can form appropriate 

 H bonds with the bases in the added DNA. 

 In other words, in an extensive synthesis, 

 pre-existing DNA should serve as a template 

 for the synthesis of complementary DNA 

 strands. Figure 21-4 shows some pyrimi- 

 dine and purine bases which do not naturally 

 or frequently occur in DNA as well as the 

 four principal types which do. The un- 

 natural or infrequent bases include: uracil 

 and 5-bromo uracil (both of which are ex- 

 pected to have the same H-bonding capaci- 

 ties as thymine); 5-methyl cytosine and 5- 

 bromo cytosine (both of which are expected 

 to have the same H-bonding capacities as 

 cytosine); and hypoxanthine (which has two 

 of the three H-bonding sites found in gua- 

 nine). If A in the single-strand preformed 

 DNA dictates its complement — by specify- 

 ing that the complementary base is a pvri mi- 

 dine that provides the proper sites for H- 

 bonding A — then one would expect thai 

 uracil or 5-bromo (or 5-fluoro) uracil can 

 substitute for the thymine in thymidine 5'- 

 triphosphate. 



When the substrate used contains do- 



