To begin, extract DNA from the plant tissue and determine the DNA concentration. Set up the reaction after adding all the required components to a 0.2 milliliter centrifuge tube. After the primer amplification, load three microliters of the PCR product and two microliters of a 5, 000 base pair DNA marker to the wells of an agarose gel.
Set the voltage to 120 volts, the current to 400 milliamps, and run the electrophoresis for 40 minutes. Use a versatile gel image analysis system to view and analyze the gel results. After running the sample through a sequencing unit, select Create a New Project and click Okay to go to the software homepage.
Under the File menu, select Import and Add Samples. Choose the sequence trace format file and import it as the file to be processed under unassembled samples. Choose Contig.
Then go to Advanced Assembly and select Assemble in Groups. When the dialogue prompting to define names appears, modify the file name in the define sample name parts section, and click Assemble to display the aligned sequence. Select Go, then choose Search Sequence and click Okay to find the matching sequence.
Choose Contig, then select Delete to remove the 5.8S and 28S regions, along with any low quality sequences. Export the output with the spliced sequences for matching. Select a BLAST search using the Nucleotide Database at NCBI.
Choose the species identification tool and the specific primer ITS-2, corresponding to the Global Pharmacopoeia Genome Database. Download sequences of three species of Angelica and one species of Peucedanum praeruptorum as an outgroup from NCBI. Analyze these sequences along with the result sequences obtained.
Then click File. Select Open a File or Session to import the file and a dialogue will appear. Choose Align followed by DNA and select Align by ClustalW for sequence comparison.
Navigate to phylogeny and click Construct Test Neighbor Joining Tree to generate a phylogenetic tree. Gel electrophoresis results showed single clear bands around 500 base pairs for samples AS1, AS2, and AS3, with no bands in the negative control, indicating successful amplification of the extracted DNA. Sequencing results identified Angelica sinensis with 100%sequence similarity using BLAST, matching results from the GPGD database.
In the phylogenetic analysis, the splicing sequences clustered with Angelica sinensis OR8797150.1, with a bootstrap support value of 100, proving the identification as Angelica sinensis.
