troubles with human genomic DNA amplification - (Mar/09/2006 )
I need to amplify human genomic DNA fusion gene in order to find the exact breakpoint of one of the fusion partners. I´ve used complementary DNA before and I got the clear product. Now I need to distinguish the right isoform (between two possible) according to the location of the genomic breakpoint (is it involved in exon or intron????). My product should be about 2kb, but I´m not successful in getting it, using as the same primers as for cDNA amplification. I´ve tried to optimize PCR by changing conditions: using Expand High Fidelity PCR System instead of AmpliTaq Gold DNA polymerase system, Mg concentration, template concentration, annealing temperature etc. Nothing helped. I repeately get 350bp product supposed to be non specific. Does anybody have any recommandations for me? Thanks Vraja
I suspect that your problem stems from the fact that you are using primers designed for cDNA, not genomic DNA. cDNA primers are usually intron spanning so that they don't amplify genomic DNA instead of the target cDNA. I suggest designing some genomic primers.