My research seeks to harness the power of DNA barcoding technology to enhance the accuracy and the efficiency of medical plant identification, thereby contributing to the persuasion and the sustainable use of their valuable natural resource. DNA barcoding technology has found broader applications, such as quality control by monitoring or protect species and biodiversity assessment. High throughput grinding and amplification are combined with DNA broadcast for species identification, which has bonded the application of herb identification based on DNA barcoding and brought out the past DNA barcoding error.
Currently, there is a lack of comprehensive guidelines on how to identify medical plants, and our process can provide detailed references. The primary research question for our laboratory in the future, where folks integrating DNA barcoding technology with technologies in our fields for broadening application and technology innovation. To begin, take 100 milligrams of fresh tissue or 20 milligrams of air-dried tissue and place it in a two milliliter centrifuge tube.
Place two five millimeter stainless steel beads into the centrifuge tube, and transfer it to a high-throughput tissue grinder, operating at 60 hertz for 60 seconds. For DNA extraction, add 800 microliters of pre-cooled nuclear isolation buffer to the sample, and place it on a vortex mixer for five minutes. Then, place the sample in a centrifuge and spin at 13, 780G for 10 minutes.
After centrifugation, remove the supernatant carefully. Next, add 600 microliters of buffer P1, followed by five microliters of RNase A to the sample. After vortexing, incubate the samples in a water bath at 65 degrees Celsius for 1.5 hours, inverting the tubes two to three times during incubation.
Then, add 200 microliters of buffer P2, mix thoroughly, and place the sample on ice for 15 minutes. Afterward, centrifuge at 13, 780G for 10 minutes. Carefully aspirate the supernatant onto a filter column AF, and centrifuge it at 13, 780G for two minutes.
Transfer the lower filtrate to a new 1.5 milliliter centrifuge tube. After calculating the volume of the filtrate, add 1.5 times the volume of buffer P3 to the sample and shake immediately to mix the sample. Add 650 microliters of the prepared mixture to an absorption column and incubate for five minutes.
Then, centrifuge at 13, 780G for 30 to 60 seconds. Add the obtained filtrate into the absorption column again. Centrifuge and discard the waste liquid.
Wash the column twice with 600 microliters of rinse solution WB.After centrifugation, discard the waste liquid, then, place the absorption column into an empty collection tube. After centrifuging the sample at 13, 780G for two minutes, leave it at room temperature to evaporate residual ethanol. Transfer the absorption column into a clean 1.5 milliliter centrifuge tube, and add 45 microliters of sterilized deionized water preheated to 65 degrees Celsius to the center of the adsorbent membrane.
Five minutes later, centrifuge the tube at 13, 780G for two minutes at room temperature. Then, use a spectrophotometer. Determine the DNA concentration in the filtered solution.
The absorbance ratios of OD260 to OD280 ranged from 1.8 to 1.84, and the DNA quantity exceeded 100 nanogram per microliter, confirming good quality of the extracted DNA 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 OK 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 Then go to Advanced Assembly and select Assemble in Groups. When the dialog prompting to define names appears, modify the file name in the define sample name parts section, and click Assemble to display the align sequence.
Select Go, then choose Search Sequence, and click OK to find the matching sequence. Choose 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 ITS2 corresponding to the global Pharmacopeia 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 dialog will appear. Choose a line followed by DNA and select a line 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 OR879715.1, with a bootstrap support value of 100, proving the identification as Angelica sinensis.
