dna extraction (6)

12368336463?profile=RESIZE_400xThe European Commission's Joint Research Centre (JRC) has published guidance on the selection and use of DNA extraction methods.

Extracting DNA of suitable quality and quantity from a test sample is a fundamental upstream step that underpins the confidence in a number of downstream analytical molecular biology based methods (e.g., qPCR. dPCR, NGS, etc.,).

This official guidance document provides advice on the selection and use of fit for purpose DNA extraction methods. Whilst this guidance uses the example of DNA extraction in the context of official controls for the analysis of genetically modified organisms (GMOs), the principles it describes are universally applicable to all DNA based methods including those for food authenticity.

Advice is provided on the selection of different protocols and decision support systems, and guidance provided on validation approaches and the assessment of DNA quality parameters, further illustrated with practical examples/solutions based on extensive collective experiences.

Access guidance: DOI: 10.2760/76162 (online)

This guidance has also been added to the Quality section of this website.

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The quality and quantity of the extracted DNA are two key aspects for a successful PCR (Polymerase Chain Reaction) amplification. Also, a reduction in time and cost required for DNA extraction are important. The aim of this study was to compare and optimise the performance of five different DNA extraction methods by boiling meat tissues from cattle, buffalo, sheep, goat, chicken, camel, horse and dog in PBS (Phosphate Buffer Saline), distilled water, alkaline lysis buffers 1, 2 or 3. The results indicated that the boiling of meat and its products in alkaline lysis buffers was the best method to extract crude DNA. The optimised crude DNA extraction protocol was coupled with PCR-RFLP (Restriction Fragment Length Polymorphism) analysis for meat species identification. The developed assay was tested on 53 commercial beef and mutton samples, out of which three samples were found to be adulterated.  

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Droplet digital polymerase chain reation (ddPCR) technology is a PCR method utilising a water-oil emulsion droplet system, where each nanoliter-sized droplet in the emulsion contains the template DNA molecules, essentially serving the same function as individual test tubes or wells in a plate in which the PCR reaction takes place. In this study, ddPCR was used to detect adulteration of acacia honey with canola (rapeseed) honey. DNA extraction from pollen in acacia honey and canola honey was performed using four different pollen treatment methods. A duplex ddPCR method was developed based on the specific target gene in acacia and canola, which permitted detecting up to 1% adulteration of canola in acacia. This method is more rapid and accurate than the accepted microscopy examination of honey pollen, but does not address exogenous sugar adulteration of honey.

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DNA authentication of wines is challenging given the acidic and alcoholic medium of wines coupled with their long storage. Russian researchers have investigated using the centrifuged debris precipitated from either red or white wines using various precipitators and co-precipitators as a source of DNA for grape varietal identification. The strategy for identification was based on direct sequencing of the PCR (polymerase chain reaction) products amplified using primers based on the grape UFGT gene locus. Although DNA extracted directly from grape varieties gave good varietal identification, there were some problems in identifcation encountered in analysing commercial wines, which indicated further research is necessary for method improvement. 

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Fruit content and fruit authenticity are crucial to informing the consumer about the quality and authenticity of jam. However, jam is one of the most difficult products to extract undegraded DNA that can be amplified from the fruit ingredients being an acid high sugar product. Czech researchers have tested three extraction methods - two commercial kits and the CTAB method on 14 different jams to find which one produced the most well-amplifiable DNA. 

       Read the full article at: DNA extraction of jam

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