Principles of Nucleic Acid Separation

Finally, the reaction mix may contain glycerol for formamide to enhance specificity (Cha et al., 1992 Sarkar et al., 1990), as well as gelatin, Triton X-100, or bovine serum albumin to stabilize the polymerase, and mineral oil to prevent water evaporation during the reaction. 

The typical PCR run (Fig. 7.1) consists of three cycles denaturation (1-2 min at 3 94°C), primer annealing (1-2 min at 50-55°C), and extension (1-2 min at 72°C). This design also requires optimization for each particular PCR. The desired blunt-ended duplex product does not appear until after the third cycle, whereupon it accumulates exponentially in subsequent cycles. The number of cycles required will depend on the efficiency of the reaction per cycle. Once the desired product has reached about 1012 copies, PCR efficiency drops significantly, and product stops amassing exponentially. This is the plateau phase continuing PCR beyond this point often results in contaminating by-products rather than more product (Cha and Thilly, 1993). 

The separation of nucleic acids by CE has become a steadily growing area of interest, especially since the inception of the Human Genome Initiative. This interest originally stemmed from the use of polyacrylamide (PA) or agarose slab gel electrophoresis as the accepted standard for nucleic acid separation (Stellwagen, 1987). 

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