PCR-based quantitation of low levels of HIV-1 DNA by using an external standard

Beginning with 10(3)-10(5) molecules of a purified HIV-1 target sequence as a starting template, we have examined the effects of starting template concentration and cycle number on the amplification efficiency of the polymerase chain reaction. An external standard DNA sequence has been designed that when added to a DNA sample enables a determination of the starting concentration of HIV-1 target sequence in that sample of DNA. Varying ratios of external standard and target DNA sequences were amplified for 22 cycles. When the starting concentration of the external standard was within 50-fold of the starting concentration of the target, the amplifications of both sequences were proportional. These same results were obtained when the two templates were amplified in the presence of an excess of heterogeneous genomic DNA. Using this quantitative method, the number of starting target molecules in a DNA sample can be calculated to within a two-fold range of accuracy.