do plasmids amplify more efficiently than genomic DNA? - (Apr/26/2006 )
I made standard curves by amplifying serial diluted plasmids and human genomic DNA. The experiment was repeated with another human genomic DNA. In both cases, there was a difference in the efficiency of the standard curves between plasmids and human genomic DNA. Plasmids amplify with an efficiency of 1.9 and human genomic DNA amplifies with an efficiency of 1.7 . How can this be? Both DNA variants contain the same target, amplified with the same primers.
All publications I read tell, that linearized plasmids are the prefered standards for quantifying. Has anybody experienced similar problems like me?
I'm not positive, but I think I may have an explanation...
think about secondary structure
your gDNA should be relatively intact if it to be an effective template. this means that it is not just a little bit of contiguous DNA from the plasmid, there is a great deal of non-target DNA in the sample, that might alter the primer-binding configurations and the overall availability of your sequence
anyways, I think that linear plasmid will be best because it is most like the cDNA you are using as template, smaller linear pieces of DNA with readily accessible target sequence
Thanks for your answer,
I will quantify genomic DNA samples. So I won't use plasmids any more. But I also wanted to know the reason for this phenomenon. So I restricted gDNA with Alu I, so that my target on the gDNA wasn't affected but the chromosomal DNA was fragmented in small pieces. But the efficiency of this fragmented DNA was the same as the not restricted. I've no idea, what happens in my PCR.
i think it's because you may have les specificity in genomic DNA than inn plasmids. That increase the complexity of fragment families. Plasmid DNA are smaller and always the same...