help with identifying deletions in tissues - (Jan/29/2008 )

I have some tumor samples and their "normal" matched tissue controls embedded in paraffin. I am looking for deletions of a particular locus on the X chromosome from male samples. I have isolated genomic DNA and have been trying semiquantitative PCR by amplifying my region of interest and comparing it with house keeping genes such as GAPDH. I'm also doing qPCR with a Light cycler, but the results don't really make sense. Is their a better way or a sure-fire way? I'm not in a genomics lab, so our knowledge of genotyping is limited, so any suggestions would be appreciated.

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Are you looking for a complete or partial deletion of an entire gene/chromosome region. If the former, you could sequence the PCR product to see if the gene of interest is identical/similar or if the latter you could make an FISH probe for the locus you're interested in.

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I'm not entirely sure if semiquantitative PCR will give you an appropriate answer. If your deletion is fairly small, you may still amplify a product, and detect it using a lightcycler with equal abundance. I think that a better approach would be to sequence PCR products like JAH suggested, or do southern blots. Cut your genomic DNA with a couple of different enzymes, and use several different probes around the area of your locus. If you have deletions in this area, you should detect shifts in the bands. Then you can use a restriction map to determine where your deletions are approximately located and do PCR to sequence them.

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According to the literature, i'm looking for both, there are cases with deletion of a large locus around my gene, as well as deletions in the N and C-termini. I figured FISH was the best method, but i'm unfamiliar with it. But i'll give both of your suggestions a go. Thanks!

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