POSTED ON BEHALF OF LIJO JOHN
Dear Forum Participants,
Further to the closing remarks made at the end of this discussion, I am pleased to provide you with a brief summary of the information shared during this discussion.
The aim of this discussion was to share practical experiences and knowledge on sample preparation from different matrices including considerations for troubleshooting.
A few resources were made available describing methods and techniques for extracting DNA from difficult samples, specifically honey and olive oil.
Both these types of sample matrices are challenging to work with because they contain very small amounts of DNA. Moreover, the quality of the little DNA that is present may be compromised. DNA, being a water soluble molecule, primarily remains in the aqueous waste portion following the processing of olives into oil. The processing steps may also cause DNA that remains in the oil to degrade. Honey, on the other hand, contains DNA in the form of pollen that may have carried over from the plants from which the nectar was obtained by the bees. Pollen is found at concentrations in the range of 1000 pollen grains/gram honey, which is relatively low and only a limited amount of DNA can be extracted from such a sample.
The articles shared during the discussion described several different choices of methods that have been demonstrated to work efficiently on these difficult sample matrices. The methods included several preparatory steps to facilitate the subsequent extraction procedure, such as, centrifugation, addition of proteases and filtration. With regards to the subsequent extraction techniques several were tested, such as CTAB and phenol/chloroform and the use of a variety of DNA extraction kits some of which were specifically designed for difficult samples.
Other approaches that may be explored to facilitate the detection of DNA from such samples is at the level of primer design. Since the DNA may be of low quality and somewhat degraded due to long storage time, the use of small amplicons would be more useful. Furthermore, the use of nested PCR as an amplification strategy may be considered in order to target low concentrations of DNA.
Additional references describing methods to extract DNA from such samples can be found below.
DNA Extraction from honey and pollen – CTAB. Available at: http://gmo-crl.jrc.ec.europa.eu/doc/Annex1-Standard%20Operating%20Procedure-extraction_GM-pollen_DNA.pdf
DNA Barcoding for Honey Biodiversity. Available at: http://www.mdpi.com/1424-2818/2/4/610/htm
Random Amplified Polymorphic DNA Analysis of Olive (Olea europaea L.) Cultivars. Available at: http://journal.ashspublications.org/content/120/3/538.full.pdf+html
Detection of genetically modified soybean DNA in refined vegetable oils. Available at: https://link.springer.com/article/10.1007/s00217-010-1238-2
Monitoring genetically modified soybean along the industrial soybean oil extraction and refining processes by polymerase chain reaction techniques. Available at: http://www.sciencedirect.com/science/article/pii/S0963996909003202
I would like to thank all who have contributed to the discussion and hope that the information shared here is useful. I look forward to further collaborations with you in the future.
posted on 2017-08-21 13:40 UTC by Dina Abdelhakim, SCBD