Search of Medline and Google Scholar using the terms “limiting
dilution” and “PCR” showed that the number of publications using
limiting dilution PCR increased to a peak of 12 publications per
year in 1999. The publications were chiefly, but not entirely, in
the areas of virology and lymphoid biology and neoplasia, presumably
because workers in these two fields were more likely
to have become aware of the technique. However between 2000
and 2002 the annual publication rate fell steeply, and publications
using limiting dilution PCR virtually disappeared thereafter. This
was undoubtedly due to publication of the method of real-time
quantitative PCR by Heid et al. [9]. Real-time PCR is a closed method
and is technically simple and these features overcome the two disadvantages
of limiting dilution PCR. Our group gratefully switched
from limiting dilution PCR to real-time PCR as soon as we became
aware of real time PCR.