Below is an example of our Certificate of Analysis for species identification and Clean Screen Next Generation Sequencing testing services.
- DNA Extraction. Using sterile techniques, a random sample of approx. 25-100 mg is homogenized using a mechanical grinding device or mortar and pestle, if necessary. Genomic DNA is extracted from the sample using the appropriate buffers for the particular matrix. An extraction negative control is included in each run to test for contamination.
- PCR Amplification. The extracted DNA is then added to a mix of enzymes, water, and buffers, along with primers designed to target and amplify specific gene regions from either the nuclear or chloroplast genome. Gene regions are carefully selected to ensure their specificity for each plant species. Regions may be universal (i.e. amplify all plants) or group-specific, depending on the nature of the material and by customer request. Through heating and cooling the sample using a Polymerase Chain Reaction (PCR), or Thermal Cycler machine, multiple copies of the genes are created for sequencing and analysis. For each PCR run, the extraction negative and water control are included to test for contamination, along with a positive control to ensure a successful PCR reaction.
- Gel Electrophoresis. In order to examine if the gene was successfully amplified, a small sample of the PCR product is stained using florescent dye and run on a gel using electrophoresis. The gel is visualized to examine the presence or absence of bands, which contain the amplified DNA. All positive PCR products are then sequenced as described below, using Next Generation Sequencing.
- Next Generation Sequencing- All positive PCR products are sequenced using a Next Generation Sequencing technology utilizing ion semiconductors. This technology is based on the detection of hydrogen ions that are released during the polymerization of DNA and synthesizes a complementary DNA strand to the sample DNA. This allows us to massively sequence in parallel tens of thousands of sequences from a sample.
- Data Analysis. DNA sequences added to a database or matrix containing authenticated reference sequences, which we have obtained from herbarium specimens (vouchers of the whole plant) or from peer-reviewed publications. Comparison of the test sample to these reference sequences through visual comparison, as well as through a phylogenetic analysis (i.e. branching diagram) is performed to identify the sample. Certificates of Analysis (CoA) are generated that summarize the data, including the species identified and the number of sequences identified per species.**Please note that this is not a quantitative test and is not representative of the weight or volume of each species in the starting material.