Limiting dilution polymerase chain reaction

Quantitation of polymerase chain reaction (PCR) targets by limiting dilution analysis requires optimization of the PCR so that it is all-or-none, replicate amplifications around the limit of dilution a which reactions commence at 0-1 targets, and calculation of target numbers using Poisson statistics. Limiting dilution PCR was used to quantitate low numbers of leukaemic cells. The amplifiable target of the leukaemic cells was the rearranged immunoglobulin gene and the amplifiable target from the total cell population was the N-ras gene. Leukaemia was quantitated at concentrations of 6.7 x 10-2 to 9.9 x 10-7•
Quantitation of DNA targets by limiting dilution is relatively straightforward. Quantitation of RNA targets is conceptually similar but raises issues of RNA degradation, efficient reverse transcription and the use of the appropriate internal and external controls.