i have a question about sequencing a mutant gene that can only be maintained by a balancer. the problem is that homozygous mutant animals are very early lethal (no embryos) so we are unable to get -/- animals and are only able to have balancer/-. how do we sequence this? if we pcr balanced stocks we will obviously have both wildtype and mutant copies and cannot get mutant copies alone. i hope that’s clear.
kris
look for double peaks in the sequence.
Clone the PCR products and sequence a number of clones. Ideally, half will be balancer and half will be mutant.
We sequence from PCR products derived from balanced lethal strains frequently.
The double peak on the ABI trace is easy to spot. We have only done this with
Translocation balanced strains, but I see no reason it won’t work well with others.
It is likely best for picking up base changes, rearrangements and such might be
more complicated. If they are suspected, I suggest using an oligo-nucleotide array
chip.
Dave
I’ve had a similar problem sequencing from RT-PCR with multiple products and solved it one of two ways: 1) clone PCR products into a Topo vector and sequence from there. 2) run an agarose gel of your PCR product, gel extract your band of interest and sequence the DNA from the extracted bands. Load the lanes with as much PCR product as possible, use multiple lanes (or PCR reactions) if needed and pool the extracted bands so you have enough DNA to sequence. Some people might think this is not good practice but I have never had a problem doing this.