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Southern blot for deletion detection....HELP!!!!!


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#1 jayminm

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Posted 09 January 2010 - 10:35 AM

I am a novice in southern blot hybridisation technique. Recently my lab detected a deletion of an entire exon. Now,my task is to show that it has actually got deleted using a Southern blot. In other words, I have to show on a gel that this particular exon is absent in the patient. How do I do that???
How do I design this expt???

#2 bob1

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Posted 10 January 2010 - 03:07 PM

design probes that outside the (deleted) exonic region and use a restriction enzyme that will give you different size bands depending on the if you have a deletion or not.

#3 jayminm

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Posted 10 January 2010 - 10:57 PM

OK! If I am right in understanding your post, you meant the foll.:
If my exon 2 has been deleted, I must design a probe which will cover exon 1 and exon 3. Then, have a restriction site which will cut between exon 1 and 3??

#4 bob1

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Posted 11 January 2010 - 03:45 PM

Like this...


Normal gene:

----------AAAAXAAAAA------------------BBBBBBBBBBBB---------------------CCCCCCCCCXCCCCCCC-----------

Mutant:

_______AAAAXAAAAA------------------------------------CCCCCCCCCCXCCCCCCC---------

Where A, B, C are exons. X = restriction site. bolded and underlined = probe site. Probe and restriction sites can be anywhere in the sequence, so long as the probe is outside the deleted region and within the restriction fragment and the RE sites are on either side of the deleted region. Deleted exon will give you a shorter fragment, thus a band lower down on the gel compared to the normal sequence.




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