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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