how to distinguish two allele? - (Oct/10/2003 )

Hello,
I have found two mutation in the same gene in a patient and I want to know if these two mutations are in the same allele or on two different ones.
Please how can I do???
I've tried to clone the PCR fragment but then the sequence was eterozygotic AAGGHH

I'm not sure that i get your question correctly.
If the loci of the mutations are close enough to be on the same PCR fragment it should be easy. I gather from your sequence that the loci are next to each other and that both of them are either an A,T or C.
In that case i can think of 2 things that could be wrong. Either the sequencing did not work well and you would have to resequence or the DNA in the sequencing reaction comes from more than one clone. If you want to make sure that the DNA is from a single clone (so from one PCR molecule) you should reclone the DNA.
If your bacteria have say a competency of 10 to the power 8 you should dilute your DNA (which is at a concentration of say 1ug/ul) 1000.000 X to get ~ 10-100 free clones with only one plasmid molecule introduced per bacteria. Pick a few clearly seperate clones and sequence those.

Hope this is usefull to you

Thank you for your kind replay

No worries, hope it will help you out solving your problem