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Using RT-PCR to find the presence of a deletion in a gene


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#1 itaimo

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Posted 13 March 2013 - 02:39 AM

Hi,

I have genomic DNA samples that some might have a deletion in a specific area in a gene and some not. and i have primers designed to that specific area without presence of the deletion.

Is it possible to check the samples for the presence of the deletion with that primer ?
what should i expect to find? when there is a sample with the deletion, the primer that is design to the amplify the sequence when there is no deletion, will not work ?
It could be possible that the deletion is in one allele only, so in that case i will have only low expression ?

How can i normalize the results? i have to find a housekeeping primer that is working with gDNA, maybe to design the common ones like Actin and GAPDH without the exon - exon junction.

#2 GNANA

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Posted 13 March 2013 - 03:45 AM

since you say deletion prone region is specific and if it is also small and distinguishable in terms of its size by PCR, how about designing primer covering few hundred base pairs at the 5 and 3' end of ur deletion region(which shd present both in wild type and the deletion), so that you can distinguish the presence of deletion by size, in this case even if the deletion is in one allele you shd get two products with size corresponding to deletion and wild type.
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#3 leelee

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Posted 13 March 2013 - 05:03 AM

Gnana's idea is a better one.

Using your current primers you will basically be expecting the PCR to fail in the case of a deletion. Having a negative PCR as your screening method is a bad idea. You will never be able to know if the PCR failed because of the deletion, or because of other factors with your PCR. Even good controls won't alleviate this, as when you are using individual, unique samples you will never know if something to do with that particular sample (e.g. inhibitors etc) are what is causing your failure.




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