I've lost my introns, can you help me find them?
April 5, 2006 12:51 PM Subscribe
Calling all biologists - I've amplified a 700 bp region of cDNA for the gene PGI and now am trying to amplify this region using genomic DNA. Way more detail inside...
posted by a22lamia to science & nature (25 answers total)
Relevant background: PGI in plants appears to have 21 introns found in conserved locations, throughout the plant kingdom. My goal is to design primers to amplify some of these introns.
So far I have amplified this 700bp region in cDNA using degenerate primers. With the sequence I got from the cDNA I designed species specific primers around the intron boundaries. Using these species specific primers I have tried amplifying this region using genomic DNA. The first month of amplifications each time I amplified the genomic DNA I would run the product out on a gel and every sucessful amplification would show a smeared band the same size as was found in the cDNA. I was also having a bit of an issue with negative controls during this time and both my advisor and I were convinced I had contaminated something with cDNA. I threw out all of my reagents and primers and started all over again designing new primers based on the cDNA sequence data. Using genomic DNA that had never been opened at the same time as cDNA was being using I ran a gradient pcr reaction with my new primers. Yet again I am getting bands the same size as my cDNA, but this time they are not smeary at all, and further, my negative controls are perfectly clear. I've picked the brains of all of my labmates and haven't come up with any reasonable solution and my advisor is out of town for another two weeks. Does anyone have an idea about what could be going on? Could I be amplifying mRNA (I can't imagine it wouldn't have degraded in the DNA isolation process)? Has anyone ever heard of a gene losing all of its introns (losing 21 seems virtually impossible)? Can anyone think of some experiments I could do to identify what I am amplifying?