Dynamic Flux Amplification (DFA)– The Next Great Nucleic Acid Diagnostic Technique
The real-time PCR rage is still afoot, and in some respects it is expanding rapidly, yet still there are so many research and diagnostic labs that have maintained their stance on probes versus double stranded DNA binding dyes. Their stance is this, double stranded DNA binding dyes are much cheaper to use on an ongoing basis than are probes.
I certainly wouldn’t disagree with this notion, that would be foolish, it is true that a great SYBR Green I assay that doesn’t produce non-specific products is less expensive per reaction. Though the reality of a double stranded DNA binding dye based assay that uses standard PCR thermal cycling conditions is going to end up producing non-specific products which are going to either have to be systematically ‘ignored’ during the amplification or they are ‘hidden’ by foreshortening the thermal cycling protocol to something less than 30 cycles (for a robust amplification protocol). Either way the non-specific products are there they are just removed from the analysis. This often results in those situtations where low copy number template containing reactions are missed and called either ‘unknown’ or worse yet ‘negative’.
In the summer of 2004 I set my mind to solving this problem with the intent of eliminating it altogether. What I came up with, with no small effort from my laboratory staff was a technique of DNA and RNA amplification that we have dubbed Dynamic Flux Amplification (DFA). It is just what the name says, a method of dynamically taking advantage of the nature ‘breathing’ , or flux, of nucleic acids. That natural breathing opens and closes regions of the nucleic acid and we target those opened regions with target specific primers so that only those opened and single stranded regions of DNA are interrogated by the primers. This, in turn, makes it so the amplification is wholly specific and subsequently the formation of non-specific products (NSP) is eliminated.
We have evaluated the DFA technique against a variety of DNA templates, as one critic asked if this were something that could only work with specific G+C content templates, and determined that even over a broad range of G+C content templates from 38-60% the DFA technique performed comparably in sensitivity to PCR and without the formation of NSPs.
The really wonderful feature of DFA that comes out of the elimination of NSPs is that a whole slew of detection techniques are opened up the diagnostician. DFA has been adapted to the following detection techniques: real-time PCR (dsDNA binding dye), gel-electrophoretic, chemiluminescent, colorimetric, and ELISA. The list is continuing to expand, and we at Signal Diagnostics are excited and thrilled to work with any customer that has a dsDNA detection chemistry that they want to test with DFA.
Give us a call at 801-580-0490 and ask for me, Brian Caplin. I want to hear from you and together we can bring a new and even less expensive diagnostic chemistry online for your DNA or RNA assay.