[Drosophila] Re: Dros Digest, Vol 89, Issue 10

Slawek Bartoszewski via dros%40net.bio.net (by slawek from univ.rzeszow.pl)
Sun Sep 16 14:35:10 EST 2012


Dear Gae,

Your explanation suggests that, the gene is not expressed...  If it is 
expressed, then clones should be available from the cDNA genome project 
- check for appropriate section in the genome annotations.  If no clone 
is described (but it suggests poor expression) you might try to use 
genomic fragment for making the probe, the intron should not interfere 
with the experiments.

Good luck,
Slawek Bartoszewski.

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>     1. Problem with Reverse Transcription PCR (kovalick_g from utpb.edu)
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> ----------------------------------------------------------------------
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> Message: 1
> Date: Fri, 14 Sep 2012 16:45:15 -0500 (CDT)
> From: <kovalick_g from utpb.edu>
> Subject: [Drosophila] Problem with Reverse Transcription PCR
> To: Dros mailing list <dros from magpie.bio.indiana.edu>
> Message-ID: <201209142145.001714 from mirapoint.utpb.edu>
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> I've been having a problem with Reverse Transcription PCR (RT PCR) that I haven't encountered before, and I am hoping I can get some advice as to how to proceed.  My goal is to clone gene fragments and use them as templates to produce probes for in situ hybridization.  Before I go to the trouble of cloning these fragments, I want to use RT PCR as a quick way of verifying that the genes are expressed during the developmental stage I am interested in (24 h adults).
>
> I designed primers to flank an intron in a gene of interest, so that a 700 bp fragment should be amplified from genomic DNA and a 600 bp fragment from cDNA.  We optimized the PCR conditions (MgCl2, cycle number, and annealing temperature) using genomic DNA and got very robust amplification of a 700 bp fragment.  However, when we tried to amplify a fragment from adult cDNA, we got very little to no amplification.  This was puzzling, since the gene in question should be expressed at moderate to high levels at all developmental stages, including the adult.  We have repeated the PCR using several different cDNA preps, including those prepared with RNA from other stages of development (0-12 h embryos, wandering-stage larvae, 24 h pupae).  The results are always the same:  Little-to-no amplification.  When we can visualize an amplification product on a gel, it is of the appropriate size (600 bp) for a product amplified from cDNA.  Our RT minus and water-only controls are negative.!
>     Our positive controls use genomic DNA as the template, and they always produce a very nice 700 bp fragment.
>
> The problem seems to be specific to this gene.  We have used the same preps to amplify cDNAs from a dozen other genes, with great success.  I rechecked the primer sequences and locations, and they seem to be OK.
>
> Does anyone have any suggestions as to why amplification works when the template is genomic DNA but not when it is cDNA?  Should I design new primers and try to amplify a different part of the gene?  Could this problem be due to secondary structure in the transcript or cDNA?  If so, is there any way to test for this?  Is there any way to reduce secondary structure during cDNA synthesis/PCR?
>
> I would appreciate any suggestions from my colleagues.
>
> Gae Kovalick
> Associate Professor
> Biology
> University of Texas of the Permian Basin
> 4901 East University Blvd
> Odessa, TX  79762
>
> Email: kovalick_g from utpb.edu
> Phone: 432-552-2265
> Fax: 432-552-3230	
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> End of Dros Digest, Vol 89, Issue 10
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