(Modified
from Till BJ, et al. 2003, Genome Res.
13:524-530)
The general
procedure is as follows:
A population is mutagenised
with EMS (or another mutagen), and
allowed to grow up and produce progeny.
DNA is isolated from the
progeny of the mutagenised individuals
Then the DNA samples are
pooled to reduce the number of PCR reactions necessary for the
detection of a mutation.
Next the pooled DNA is
amplified using fluorescently-tagged, gene-specific primers.
These PCR products are
subsequently digested with Celery Juice Extract (CJE) (Till
BJ, et al., 2004 Nucleic Acids Res.
32:2632-2641.), which contains an endonuclease that
specifically cleaves at the single base pair mismatches that
result from heterozygous point mutations, or polymorphisms.
The digested DNA is run
on a denaturing, polyacrylamide Li-cor gel in order to
identify DNA populations that carry a mutation in the gene
being tested. The position of the mutation within the
amplicon can be calculated from the size of the
complementary bands labelled with the 3' and 5' fluorescent
tags.
Once a mutation has been
identified the procedure is repeated using DNA from
individuals that make up the pooled population that
exhibited the mutation in the primary screen.
