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Low copy numbers of DNA or RNA sequences can be quantified easily by combining PCR (Polymerase Chain Reaction) and TGGE (Temperature Gradient Gel Electrophoresis). This combination allows to overcome the problems of quantitive PCR caused by plateau effects, uneven priming or, variable cycle efficiencies. We have developed a general method using an internal standard identical, except for a single nucleotide, with the authentic template. After PCR, the amplified nucleic acids, although differing only in one single base position, can be separated by TGGE (1, 2). Fig. 1 shows a generally applicable protocol. Because of identical priming sites and almost identical sequences in between, the ratio between template and standard remains constant during extensive PCR cycling. Subsequently, a small amount of labelled standard (approx. 1 ng) is added to the 0.1 1 Hg of amplified DNA. After denaturation and renaturation, the labelled standard has formed homoduplexes with amplified standard and heteroduplexes with amplified template. The ratio is identical with that of standard and template before amplification. An aliquot of this mixture is analyzed in the TGGE gel with the temperature gradient being superimposed on the electrical field in parallel. The amplified standard as a homoduplex exerts the highest thermal stability and, therefore, migrates to the highest temperature in the T-gradient. The heteroduplex denatures partially at lower temperatures due to the mismatch formation. Thus, it is retarded drastically in the gradient at lower temperature in comparison to the homoduplex.

Quantitative PCR: the determination of template copy numbers by temperature gradient gel electrophoresis (TGGE)