Quantitative Digital and Real Time PCR for GM Alfalfa

One of the advantages of digital droplet PCR (ddPCR) over that of Real Time PCR (qPCR) is that quantification of target genes can be achieved in the absence of a standard graph.  Here, we demonstrate accurate quantification of GM alfalfa in alfalfa hay samples in the absence of pure GM alfalfa plant reference material.  DNA was extracted from alfalfa hay core samples using the FASTMAG DNA purification system. A plasmid with one copy each of the GM Alfalfa target and a species specific target was used as positive control.

For quantification by ddPCR, the copy number of the GM target was normalized with the copy number of the species specific target. Samples with equal copy numbers forthe GM and the species target were calculated to be 100% GM Alfalfa by ddPCR. Using qPCR, sample values were normalized to the plasmid and gave identical results to those using ddPCR. No sample with values above 100% was found. This confirms the ability to quantify GM in the absence of an external reference using ddPCR. It also shows that the FASTMAG DNA extraction system yields highly pure DNA suitable for sensitive applications such as ddPCR .