Detection, quantification, and confirmation of point mutations in
single cells. Somatic nucleotide changes are detected first by direct
sequencing of PCR product from single cells. Mutations
(Middle) are determined as changes in the sequence
compared with the sequences from the majority of cells of the same
individual (Top). After an initial identification of a
mutation, a duplicate PCR was performed from the DNA of each presumably
mutant cell (PCR 2). The mutations then were confirmed and quantified
by one of the following methods, depending on the type of the mutation.
Note that in each case, the two duplicate experiments yielded very
similar results. mut, mutant; wt, wild type. (A) If the
mutation resulted in a nucleotide change within a restriction site (in
this example, a G → C change creates a HinfI
restriction site, G′ANTC), direct restriction analysis was used to
confirm and quantify the mutation by gel densitometry
(Bottom). (B) A deletion/insertion
within a C-tract was confirmed by a variant of the T-PCR approach (15).
Briefly, a PCR fragment was restriction-digested to provide a labeled
PCR/restriction fragment ≈50-nt long, which included the
C-tract. The DNA was run on a sequencing gel. A deletion or insertion
in the C-tract manifested itself as a band shift. The weaker subbands
do not represent mutations but rather aberrant PCR products that
ultimately limit the assay sensitivity. The image was quantified by
using a PhosphorImager (Molecular Dynamics). (C) If a
mutation did not alter a restriction site or the C-tract length, direct
sequencing of the PCR fragments was used.