DNA Barcoding: Definition, Principle, Steps

DNA barcoding is a modern molecular technique used for the identification and authentication of biological species by analyzing a short, standardized region of DNA called a DNA barcode.

In pharmacognosy, DNA barcoding is particularly useful for identifying medicinal plants and crude drugs, especially when the drug is:

• Dried
• Powdered
• Fragmented
• Difficult to identify morphologically
• Closely related to another species
• Suspected to be adulterated

Simple definition ⭐

DNA barcoding is a molecular identification technique in which a short, standardized DNA sequence is used to identify a species.

Why is DNA Barcoding Needed?

Traditional identification of medicinal plants is mainly based on:

• Morphology
• Organoleptic characters
• Microscopy
• Chemical constituents

However, these methods can become difficult when the plant material is powdered, processed, or highly fragmented.

For example, two plant species may look very similar but have different DNA sequences.

DNA barcoding can help distinguish between them.

Principle of DNA Barcoding

The basic principle is:

Different species have characteristic DNA sequences in specific regions of their genome.

A particular short DNA region is selected as a barcode region.

The DNA from the unknown sample is:

Extracted → Amplified → Sequenced → Compared with reference sequences → Species identified

Steps of DNA Barcoding

Step 1: Sample Collection

A sample of the plant or crude drug is collected.

The sample may be:

• Fresh plant material
• Dried material
• Powdered drug
• Herbal formulation

Step 2: DNA Extraction

DNA is isolated from the sample. The extracted DNA should be of sufficient quality and quantity for further analysis.

Step 3: PCR Amplification

The selected DNA barcode region is amplified using Polymerase Chain Reaction (PCR).

PCR:  PCR produces millions of copies of the selected DNA region so that it can be analyzed.

Step 4: DNA Sequencing

The amplified DNA fragment is sequenced to determine its nucleotide sequence.

The sequence contains:

A – Adenine
T – Thymine
G – Guanine
C – Cytosine

Step 5: Sequence Comparison

The obtained DNA sequence is compared with sequences available in reference DNA databases. A close sequence match helps determine the identity of the species.

Step 6: Species Identification

Based on the sequence comparison, the unknown sample can be assigned to the most appropriate species.

Important DNA Barcode Regions ⭐

Different organisms require different barcode regions.

For plants, commonly used barcode regions include:

1. rbcL

The rbcL gene is located in the chloroplast genome.

Advantages:

• Relatively easy to amplify
• Good universality
• Useful for plant identification

Limitation: It may have relatively low discriminatory power between closely related species.

2. matK

matK is another chloroplast DNA barcode commonly used for plants. It generally provides better discrimination than rbcL for many plant groups.

Important:

rbcL + matK are widely used as core plant DNA barcode regions.

3. ITS / ITS2

ITS (Internal Transcribed Spacer) is a nuclear ribosomal DNA region. ITS2 is particularly useful for distinguishing many closely related medicinal plant species.

Advantages

• High discriminatory power in many groups
• Useful for medicinal plant authentication

Other regions

Depending on the plant group, other markers such as:

• trnH-psbA
• trnL-F

may also be used.

DNA Barcode vs DNA Barcoding

DNA Barcode: The short standardized DNA sequence used for identification.

DNA Barcoding: The complete process of using that DNA sequence to identify a species.

Applications in Pharmacognosy

DNA barcoding has several important applications.

1. Authentication of Medicinal Plants: It can confirm whether the plant material belongs to the claimed species.

2. Detection of Adulteration: It can identify substitute or adulterant species present in herbal drugs.This is particularly useful when the adulterant looks similar to the genuine drug.

3. Identification of Powdered Drugs: Morphological identification becomes difficult after grinding.DNA barcoding can sometimes identify the plant source even from powdered material.

4. Herbal Medicine Quality Control

It can be used as a tool for authentication and quality control of:

• Herbal drugs
• Herbal medicines
• Plant-based formulations
• Raw materials

5. Identification of Closely Related Species: Closely related species may be difficult to differentiate by morphology.DNA sequence differences can provide additional evidence for identification.

6. Detection of Substitution: A commercially supplied medicinal plant may be replaced with another species.DNA barcoding can help detect such substitution.

7. Conservation and Biodiversity Studies

DNA barcoding can also assist in:

• Species identification
• Biodiversity studies
• Conservation of medicinal plants
• Documentation of plant resources

Advantages of DNA Barcoding

1. Highly specific molecular identification method.
2. Can identify species from small biological samples.
3. Useful for powdered and fragmented materials.
4. Helps detect adulteration and substitution.
5. Does not depend entirely on external morphology.
6. Useful for closely related species.
7. Can support quality control of herbal drugs.
8. Results can be compared with reference DNA databases.
9. Useful when diagnostic morphological characters are absent.

Limitations of DNA Barcoding

DNA barcoding is powerful, but it is not a perfect method.

1. Closely related species may have very similar barcode sequences: A single barcode may not always distinguish closely related species.

2. Hybridization: Hybrid plants can make species identification more complicated.

3. Processed materials

DNA may become degraded during:

• Heating
• Extraction
• Processing
• Long-term storage

Therefore, highly processed herbal products may be difficult to analyze.

4. Contamination: DNA from another organism can contaminate the sample and affect the result.

5. Reference database limitations: Correct identification depends on the availability and quality of authenticated reference sequences.

6. It may not determine chemical quality

This is a very important point:

DNA barcoding identifies biological origin, but it does not directly tell us the concentration of active chemical constituents.

Therefore, DNA barcoding should ideally be used along with microscopic, physicochemical and chemical evaluation.

DNA Barcoding in Drug Authentication

A simple example:

Suppose a crude drug is claimed to be Plant A.

Traditional method:

Examine:

Colour → Shape → Microscopy → Chemical tests

DNA barcoding:

Sample → DNA extraction → PCR → Sequencing → Database comparison → Species identification

If the DNA sequence matches Plant B instead of Plant A, the sample may be adulterated or substituted.

DNA Barcoding vs Traditional Evaluation

Editorial Note

This article has been carefully researched and written by Deepak Rajput with a focus on accuracy, clarity, and evidence-based healthcare information. Pharmaacademias.com