TGGE in Quantitative PCR of DNA and RNA

The copy numbers of DNA sequences (templates) can be determined quantitatively by a combination of PCR and temperature gradient gel electrophoresis (TGGE; 1 ,2 ; Chapter 20 ) as illustrated in Fig. 1 . Briefly, quantitation is performed by addition of a calibrated amount of DNA “standard” to the template that is subsequently amplified by PCR. The standard is identical to the template except for a single base substitution. Because of the identical priming sites and almost identical sequence composition, both template and standard are coamplified homogeneously; the relative amounts of each product type at any time point accurately reflect the original ratio prior to amplification. This holds true for the exponential amplification phase as well as for the stationary phase.