A follow-up of "Expert advice needed on mutations/deletions"
Yong Shu Liu
liu at ariel.its.unimelb.edu.au
Tue Dec 19 01:13:31 EST 1995
Well, that was how I found the deletion. Perhaps I did not make myself
clear. I'll try now:
1. I have found the deletion by RFLP, ie cutting with enzymes.
2. What I want to know now is that the deletion occurs the ONE OR
BOTH chromosomes in one cell. Because what I have used was a piece of tumour.
After PCR reaction, I have got some wild type product which can be cleaved
by restriction enzyme. And the left-over band containing the deletion. But
the puzzle is whether the molecules carrying deletion were initially from ONE
or A GROUP of cancer cells in which BOTH chromosomes were carrying deletion
OR they were from ONE chromosome of some cells.
Any further comments? Probably in-situ allele specific PCR could help
but it is too hard to get started.
Thank you again.
On Tue, 19 Dec 1995, Dean H. Saxe wrote:
> The easiest approch would be to determine whether the deletion causes a
> restrcition site polymorphism, i.e. is a new site created or is a site
> destroyed by the mutation. Amplify the region of interest, digest with
> an appropriate restriction endonuclease and separate the fragments on a
> gel. If one of the amplified fragments is cut (i.e. contains the
> restriction site) then you should see one full length product and two
> smaller bands. This would indicate heterozygosity for the deletion
> allele. If both cut (or don't cut) then the deletion is homozygous.
> Just make sure you add enough enzyme to ensure complete cutting, patial
> cutting may skew your results.
> Hope this helps!
> Dean H. Saxe "Life gives us hurdles, and we must jump,
> dsaxe at bimcore.emory.edu and if you fall, you get your ass back up!"
> -Millan & Kenzie "Blow"
> Graduate Student, Dept. of Genetics & Molecular Biology, Emory Univ.
> My home page --- <URL: http://userwww.service.emory.edu/~dsaxe>
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