identification of intron-exon organization for gapdh - (May/21/2011 )
We have tried to identify the intron and exon sequences of gapdh gene from a frog species. Only the mRNA sequence has been identified completely (including both the UTRs) for this frog and no gene data is available.Hence, we have been using xenopus tropicalis gapdh gene info. as our reference.
We have designed various primers in the UTR regions and in the exons (identified based on xen trop data) and used them with genomic DNA as template. We had partial success with a primer in exon 6 showing amplification with a primer in 3'utr. Upstream to the exon6, we do not get any amplification- not even a non-specific product or a pseudogene is seen. I do not understand why this has been happening.
Any suggestions is most welcome.
thanks and regards,
Nirupa S Mogili
Have you used xenopus DNA as a positive control? How big are the introns expected to be? If they are very big, have you adjust your PCR conditions for long distance PCR?