DNA Testing Perspectives with Susan J. Murch, PH.D.
As Alkemist Labs begins providing Next Generation DNA testing services to clients, we are expanding our education to help demystify testing issues around using DNA for botanical identity in the natural products industry. Here we have some explanations to common questions from our friend and colleague Susan J. Murch, Ph.D., Professor of Chemistry, University of British Columbia from an interview she sent us. Some of you may have seen her stellar presentation at the AHPA meeting in Anaheim last month.
DNA burst upon the supplements scene in February 2015 when the New York AG took action against four store brands based upon DNA test results that were roundly criticized by the industry. But it raised some questions:
What about unexpected substances that were detected, like rice powder?
“DNA barcoding relies on determining the chemical composition of a small piece of the DNA. All DNA is made up of only 4 chemicals known by the letters A,G,C, and T. The sequence is a list of these letters in the order that the chemicals appear in the DNA strand. The theory of DNA barcoding is the identification of a specific short fragment of the DNA that is characteristic of the species. Ideally, one would find a short fragment that was different for each species and could be used to positively identify a species.
Each fragment is “amplified” or “copied” in order to have enough of the DNA to measure. So, if you had 1 piece of the correct sequence of rice DNA and you ran the amplification process through 50 cycles (called a thermocycler, this process makes copies of DNA), you would end up with 1125899906842620 identical pieces. You would then determine the chemical composition of these pieces as a series of A,G,C,T. Rice is most commonly identified by 2 regions named “rbcL” and “matK”. The database for the DNA barcoding projects lists 4 sequences for rice using 2 different regions. Region matK can have a length of 1186, 1378, or 1431 sequences of AGCT in different orders. Region rbcL has a length of 703
So, if you have an unknown sample and you measure a matK and rbcL region and they match the order of A,G,C and T previously found for rice, then your results are consistent with having rice in the sample.
What you don’t know and can not possibly are the following:
– how much rice is there? You did not measure quantity.
– are there any other plant species that have the same sequence in these 2 regions as rice?
– Is this short sequence really unique to rice or might it be something else? No one knows this.
– are these standards appropriate? There are only 5 sequences, all from the Philippines and we don’t know what type of rice this is, who collected it, how was it identified, where was it grown, etc. etc. etc.?”
What degree of certainty does DNA testing provide? 30 percent, 80 percent, etc.?
“It is difficult to get an idea of the overall certainty of DNA barcode testing because there are differences in accuracy at different steps as well as differences in accuracy between different regions.
The process of preparing a sample for DNA barcoding involves extracting the DNA from the sample, choosing which segment of the DNA to study, amplifying the specific fragment of DNA that is chosen as the “barcode region” and determining the chemical composition of the specific region (ie sequencing).
I have not found any studies that show recovery of the amount of DNA or the quality of the DNA recovered from manufactured natural products. The degree of certainty of methods using the matK and rbcL regions to distinguish between plant species has been reported to be 53.1% and 46.5% respectively. Overall the best estimate of certainty may be somewhere between 53% and 85% depending on the species used in different published studies. The main point that the public should know is that a DNA result is not an absolute answer.”
What are the limitations of DNA testing to identify plant extracts? Does accuracy depends on how the extract is made?
“Accuracy definitely depends upon how extracts are made. The recommendations of the Barcode of Life consortium indicate that ethanol causes problems. If the product is made from an ethanol extract that is then dried or processed in some other way, the short sequence of DNA measured can be degraded or sheared or not extracted. Therefore, the product would test negative. Likewise, products made from root material have less of the chloroplast DNA, which has most of the barcode sequences, so there were fewer fragments to start with and greater likelihood of false negative findings.”
What we can conclude from Dr. Murch’s comments is that one of the first questions you ask is which regions of the plant genome were used to determine species identity and are they appropriate? As with all testing methods, transparency as to how the results were obtained has everything to do with the validity of the results. Method validation and reliable standard reference materials are as important for DNA barcoding as for any other chemical analysis method.