Transcript Amplification in genomic DNA PCR - (Feb/22/2008 )
I'm amplifying housekeeping genes (GAPDH, ACTB and B2M) from genomic DNA using HotStart Taq Polimerase, to my surprise I always find that the band that I'm amplifying is the one that correspond to the transcript and not to genomic (ej. GAPDH amplification of 170bp correspond to transcript and 240bp to genomic because fwd primer is in exon 2 and rev primer is in exon 3). This can be a RNA contamination??
The Taq Pol can't amplify RNA...and my samples don't undergo a retro-transcription...so why I'm seeing the band without the intron? The primers were checked and don't amplify other genes.
I would really appreciate if someone can give any advice. THX
Did u get ur amplicon sequenced....and look for possible pseudogenes present...they can cause problem!!!!