Introduction to DNA barcoding technology

The current methods for identifying the identity of Chinese herbal medicine extracts or natural raw materials generally involve morphological, microscopic, and physicochemical identification. However, these methods are not precise in species differentiation, and the identification results can be easily influenced by environmental factors. In contrast, DNA identification has gained widespread popularity as it offers strong species discrimination capabilities, requires low sample quantities, and is not limited by environmental factors. OriGene’s standard DNA testing service utilizes DNA barcoding technology and optimized testing processes to accurately identify the species identity of Chinese herbal medicine or natural product raw materials for our customers. The process involves the following six main steps:

Step 1 Sample DNA Extraction

The success of DNA identification testing largely depends on the concentration and purity of the extracted DNA from the sample. DNA extraction consists of two main processes: DNA isolation and purification. DNA can be extracted from different parts of the sample, such as plant leaves, roots, animal muscle tissues, blood, etc. DNA isolation involves disrupting the cellular structure of the sample to release the DNA, followed by purification to remove impurities such as proteins, RNA, cell membranes, and others. In addition to using conventional recognized DNA extraction methods and kits, our company also optimizes the sample DNA extraction method based on the characteristics and chemical composition of the sample. This maximizes the quality of the sample DNA for subsequent identification testing, thereby increasing the success rate of the identification process.

Step 2 PCR Amplification

Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify small amounts of target DNA fragments to detectable levels. PCR consists of three main steps: denaturation, annealing, and extension. During the testing process, we will continuously optimize reaction conditions such as temperature, primer concentration, enzyme type and concentration, and utilize appropriate controls, reagents, and instruments to ensure the reliability and accuracy of the PCR reaction. After PCR amplification, the amplified PCR products will be processed and undergoes subsequent testing by our technical team.

Step 3 Gel electrophoresis

After PCR amplification, the sample DNA undergoes preliminary analysis through DNA gel electrophoresis. During gel electrophoresis, the PCR products are loaded into wells on a gel and an electric field is applied, causing the DNA molecules to move within the gel. Due to the varying mobility of DNA molecules of different sizes in response to the electric field, they migrate at different rates towards the positive electrode, resulting in distinct bands of varying sizes and positions on the gel. Technicians will visualize these bands using DNA stains and perform preliminary result analysis. Furthermore, purified DNA bands are subsequently used for DNA sequencing analysis.

Step 4 DNA Sequencing

Sanger DNA sequencing is a method used to analyze the nucleotide sequence of a target DNA fragment, which refers to the arrangement of adenine (A), thymine (T), cytosine (C), and guanine (G) bases. During the DNA sequencing process, the DNA sample is labeled, and the nucleotide sequence of the DNA fragment is determined using a sequencing instrument. Since different species have their unique genetic codes, researchers can rapidly and accurately analyze a large amount of DNA sequence data using DNA sequencing, deciphering the genetic code of species DNA. This information, in combination with DNA barcoding, helps confirm the species identity.

Step 5 DNA Sequence Alignment

After obtaining the DNA sequence of the test sample through DNA sequencing, these sequences are aligned against DNA sequence data published by other research groups in the GenBank species database using tools like BLAST (Basic Local Alignment Search Tool) or BioEdit. By comparing sequence coverage and similarity, technicians can determine the species information of the most similar DNA sequence and identify the species identity of the test sample.

Step 6 Species Identification

DNA barcoding is a method of species identification that utilizes a standardized, sufficiently variable, relatively short, easily amplifiable DNA fragment from the genome of an organism, which can represent the species. It possesses high uniqueness and repeatability. Currently recognized DNA barcodes include mitochondrial cytochrome c oxidase subunit I (COI), mitochondrial cytochrome b (CYTB), and mitochondrial 16S ribosomal RNA (16SrRNA) for animal DNA barcoding, the large unit of ribulose-bisphosphate carboxylase gene (rbcL), internal transcribed spacer 2 (ITS2), and psbA-trnH intergenic spacer (psbA-trnH) for plant DNA barcoding, and ITS2 for fungal DNA barcoding. With technological advancements, an increasing number of gene regions are being proposed as potential DNA barcodes.

Although OriGene’s standard DNA barcoding testing service can provide species identification for most common traditional Chinese medicinal herbs or natural raw materials available in the market, conventional DNA testing may not be effective for processed products (such as granules, decoctions, etc.) or for analyzing specific breeding. To address these identification challenges, our research team, with over 30 years of experience in DNA identification development, can customize the most suitable identification approach based on the characteristics of the customer’s testing samples and identification requirements. This includes optimizing the testing process and conditions, DNA extraction, PCR conditions, and standardizing the proprietary identification method for subsequent testing purposes.

For more information about OriGene’s DNA testing services, please contact our business representative.