A Review on DNA Finger Printing Techniques in Identification of Drugs of Natural Origin

Abstract

DNA fingerprinting techniques have become essential tools in the identification, authentication, and quality control of drugs of natural origin, especially herbal medicines. These techniques allow precise genetic identification by analyzing specific regions of the DNA unique to each species, ensuring the correct sourcing of plant or animal materials used in drug formulations. The major DNA-based methods include Random Amplified Polymorphic DNA (RAPD), Simple Sequence Repeats (SSR), Amplified Fragment Length Polymorphism (AFLP), and DNA barcoding. These techniques help differentiate between closely related species, detect adulterants, and confirm the authenticity of natural products, minimizing the risk of contamination or substitution. DNA fingerprinting ensures the traceability of raw materials, promotes the standardization of natural drugs, and supports regulatory frameworks for ensuring the safety, efficacy, and consistency of herbal medicines. Its application enhances the quality assurance process, making DNA-based methods indispensable for maintaining the integrity of the natural drug supply chain.

Keywords

Introduction

Natural products, particularly medicinal plants, have been used for centuries as remedies for various ailments. However, as the demand for these natural drugs has surged in recent years, concerns regarding the authenticity, purity, and quality of herbal medicines and plant-derived pharmaceuticals have emerged. Misidentification, adulteration, and contamination of natural drugs are common issues that can compromise the efficacy and safety of these products. To address these challenges, DNA fingerprinting techniques have become indispensable tools for the accurate identification and quality control of drugs of natural origin. ⁽¹⁾

DNA fingerprinting involves the analysis of specific regions of an organism's genetic material to produce a unique genetic profile or "fingerprint." This molecular approach allows for precise species identification, even when traditional methods, such as morphological or chemical analysis, are unreliable due to processing, storage, or environmental factors. Unlike conventional approaches, DNA fingerprinting can detect subtle genetic differences between closely related species, offering a high degree of accuracy in differentiating authentic medicinal plants from adulterants or substitutes. ⁽²⁾

1. Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA is a widely used DNA fingerprinting technique that amplifies random segments of genomic DNA using short, arbitrary primers in a Polymerase Chain Reaction (PCR) process. This method is especially useful in identifying genetic polymorphisms between individuals or species without prior knowledge of their genomic sequences. RAPD is commonly applied in the authentication and quality control of natural drugs, particularly herbal medicines, where it aids in differentiating closely related species and detecting adulteration. ⁽³⁾

Principle of RAPD:

RAPD relies on the amplification of random DNA segments using a single short (usually 10-mer) primer of arbitrary sequence, which binds to complementary sites across the genome. The number and size of amplified fragments depend on the distance between the primer binding sites, resulting in a polymorphic pattern that varies between individuals or species. These amplified fragments are visualized on an agarose gel, producing a unique "fingerprint" for each organism. ⁽³⁾

Procedure of RAPD:

1. DNA Extraction: Genomic DNA is isolated from the plant or drug material of interest.
2. PCR Amplification: A PCR reaction is carried out using a single short primer of arbitrary sequence. The primer binds randomly to different parts of the genome, and DNA fragments between these binding sites are amplified.
3. Gel Electrophoresis: The amplified DNA fragments are separated using agarose gel electrophoresis, which allows visualization of the polymorphic band patterns.
4. Analysis of Results: The banding patterns generated from different samples are compared to identify similarities or differences between species or individuals. The variation in these patterns reflects the genetic polymorphisms present. ⁽⁴⁾

2. Simple Sequence Repeats (SSR)

Simple Sequence Repeats (SSR), also known as microsatellites, are short tandem repeats of 1–6 nucleotide sequences found abundantly throughout the genome. These repeats are highly polymorphic due to variations in the number of repeat units between individuals or species, making SSRs highly useful as molecular markers for DNA fingerprinting. SSR markers are extensively used in the identification and authentication of drugs derived from natural sources, particularly in the standardization and quality control of herbal medicines. ⁽⁵⁾

Principle of SSR:

SSRs consist of repeating units of short DNA sequences (e.g., di-, tri-, or tetra-nucleotide repeats). The number of repeats at a given locus can vary significantly between individuals or species, generating genetic variation that can be detected through PCR amplification. By targeting these variable regions, SSR markers can produce a species-specific fingerprint, which can then be compared across samples to identify differences or confirm authenticity. ⁽⁵⁾

Procedure of SSR:

1. DNA Extraction: Genomic DNA is extracted from the natural drug material (e.g., plant or animal tissue).
2. Primer Design: Specific primers are designed to flank the SSR regions of interest. These primers target regions containing short tandem repeats.
3. PCR Amplification: The DNA surrounding the SSR regions is amplified using PCR. The resulting amplicons vary in length depending on the number of repeat units at each locus.
4. Gel Electrophoresis or Capillary Electrophoresis: The amplified products are separated by size, typically using polyacrylamide gel electrophoresis or automated capillary electrophoresis. The size of the PCR products correlates with the number of repeats at each SSR locus.
5. Data Analysis: The banding patterns or peaks are analyzed to determine the length of the repeats, allowing the identification of polymorphisms. These polymorphisms serve as molecular markers for distinguishing species or populations. ⁽⁶⁾

3. Amplified Fragment Length Polymorphism (AFLP)

Amplified Fragment Length Polymorphism (AFLP) is a powerful DNA fingerprinting technique that combines restriction enzyme digestion of DNA with selective PCR amplification of a subset of the resulting fragments. AFLP is widely used for the identification and authentication of drugs of natural origin, particularly in the botanical and pharmaceutical industries, where the ability to distinguish between species and detect genetic variability is crucial for quality control and standardization. ⁽⁷⁾

Principle of AFLP:

AFLP works by cutting the genomic DNA of an organism with two restriction enzymes, typically a frequent cutter and a rare cutter. Short double-stranded oligonucleotide adaptors are then ligated to the sticky ends of the restriction fragments, and selective PCR amplification is performed using primers complementary to the adaptors, along with selective nucleotides that amplify only a subset of the fragments. The amplified fragments are separated by gel electrophoresis or capillary electrophoresis, resulting in a polymorphic banding pattern that serves as a genetic "fingerprint" for each organism. ⁽⁷⁾

Procedure of AFLP:

1. DNA Extraction: Genomic DNA is extracted from the sample material, such as medicinal plants or animal-derived drugs.
2. Restriction Digestion: The extracted DNA is digested with two restriction enzymes: a rare cutter (e.g., EcoRI) and a frequent cutter (e.g., MseI). These enzymes produce a large number of DNA fragments with sticky ends.
3. Adaptor Ligation: Synthetic adaptors are ligated to the sticky ends of the restriction fragments. These adaptors provide binding sites for the PCR primers used in the next step.
4. Selective PCR Amplification : Primers complementary to the adaptor sequences, along with selective nucleotides, are used to amplify a subset of the restriction fragments. The selective nucleotides limit amplification to fragments with specific sequences at the restriction sites.
5. Fragment Separation and Visualization: The amplified fragments are separated by gel electrophoresis or capillary electrophoresis, generating a unique banding pattern based on fragment lengths.
6. Data Analysis: The resulting banding patterns are compared between samples to identify species, detect genetic variation, or confirm authenticity. Differences in banding patterns reflect polymorphisms at the restriction sites, making AFLP highly effective for distinguishing between species or populations. ⁽⁸⁾

4. DNA barcoding

DNA barcoding is a molecular technique used to identify and distinguish species based on short, standardized regions of their DNA. This method involves sequencing a specific region of the genome, which serves as a "barcode" for identifying species. In the context of identifying drugs of natural origin, particularly medicinal plants and herbal products, DNA barcoding has proven to be an essential tool for species authentication, quality control, and the detection of adulteration or substitution. ⁽⁹⁾

Principle of DNA Barcoding:

DNA barcoding is based on the principle that certain regions of the genome are highly conserved within a species but show sufficient variation between species. For plants, the commonly used barcode regions are from the chloroplast genome, such as the rbcL (ribulose-1,5-bisphosphate carboxylase) and matK (maturase K) genes. For animals, the COI(cytochrome oxidase I) gene in the mitochondrial genome is typically used. ⁽⁹⁾

The DNA barcode region is amplified using specific primers in a PCR reaction, and the resulting fragment is sequenced. The sequence is then compared to a reference database (such as the Barcode of Life Data Systems, or BOLD) to identify the species by matching the sequence to known species in the database. ⁽⁹⁾

Procedure of DNA Barcoding:

1. DNA Extraction: DNA is extracted from the material of interest (e.g., medicinal plants, animal products) using standard techniques.
2. PCR Amplification: A specific region of the genome (the "barcode" region) is amplified using primers that target highly conserved sequences flanking the variable region. For plants, commonly used regions include rbcL and matK; for animals, the COI gene is often used.
3. Sequencing: The amplified barcode region is sequenced to generate a unique DNA sequence for the sample.
4. Database Comparison: The obtained sequence is compared to a reference database (such as BOLD or GenBank) to identify the species based on sequence similarity.
5. Species Identification: The closest matching sequence in the database is used to identify the species. If no match is found, it may indicate that the species is not yet recorded in the database, or that the sample is adulterated. ⁽¹⁰⁾

5. Sequence Characterized Amplified Region (SCAR)

Sequence Characterized Amplified Region (SCAR) is a DNA fingerprinting technique that allows for the development of species-specific molecular markers based on the amplification of unique DNA sequences. SCAR markers are generated by converting polymorphic DNA fragments, identified from techniques like RAPD (Random Amplified Polymorphic DNA) or AFLP (Amplified Fragment Length Polymorphism), into more specific and reproducible markers. SCAR is extensively used in identifying and authenticating species in natural products, particularly medicinal plants, and ensuring quality control in the herbal medicine industry. ⁽¹¹⁾

Principle of SCAR:

The SCAR technique involves isolating a polymorphic DNA fragment from a technique like RAPD or AFLP, sequencing the fragment, and designing specific primers flanking the unique sequence. These primers amplify the sequence, creating a highly specific, reliable, and reproducible marker for that particular species or population. The development of SCAR markers improves the specificity of polymorphic markers, allowing for more accurate identification. ⁽¹¹⁾

Procedure of SCAR:

1. Polymorphic Fragment Identification (via RAPD or AFLP): A polymorphic fragment that distinguishes one species or population from another is identified using a preliminary technique like RAPD or AFLP.
2. Cloning and Sequencing of the Fragment: The polymorphic fragment is cloned and sequenced to identify the unique nucleotide sequence that serves as a genetic marker for that species.
3. Designing SCAR Primers: Based on the sequence of the polymorphic fragment, specific primers are designed to target the flanking regions of the fragment. These primers are unique to the species of interest.
4. PCR Amplification: The specific SCAR primers are used in PCR to amplify the sequence-characterized region of the genome. This amplification is highly specific to the species for which the SCAR marker was developed.
5. Gel Electrophoresis: The PCR products are analyzed through gel electrophoresis to visualize the presence or absence of the species-specific SCAR marker.
6. Data Analysis: The presence of the expected band indicates the species or population being targeted, allowing for precise species identification or authentication. ⁽¹²⁾

6. Restriction Fragment Length Polymorphism (RFLP)

Restriction Fragment Length Polymorphism (RFLP) is one of the earliest DNA fingerprinting techniques used to detect genetic variation by identifying differences in DNA sequence based on restriction enzyme cleavage patterns. RFLP works by exploiting the fact that restriction enzymes cut DNA at specific recognition sites, and variations in the DNA sequence can alter the pattern of DNA fragments generated. RFLP has been used extensively in the identification of plant and animal species, including drugs of natural origin, and remains valuable in quality control, species authentication, and genetic studies. ⁽¹³⁾

Principle of RFLP:

RFLP relies on the use of restriction enzymes, which recognize and cut specific sequences of DNA, known as restriction sites. If there is a mutation or polymorphism in or near these recognition sites, the restriction enzyme will either fail to cut or cut at an additional site, resulting in different lengths of DNA fragments. These fragments are then separated by gel electrophoresis and visualized, producing a unique banding pattern that can be compared across individuals or species. Differences in fragment sizes indicate polymorphisms in the DNA, which are used as genetic markers. ⁽¹³⁾

Procedure of RFLP:

1. DNA Extraction: DNA is extracted from the biological material, such as medicinal plants or animal-derived drugs.
2. Restriction Enzyme Digestion: The extracted DNA is treated with restriction enzymes, which cut the DNA at specific sites based on their recognition sequences.
3. Fragment Separation (Gel Electrophoresis): The resulting DNA fragments are separated by size using gel electrophoresis. Shorter fragments move faster through the gel than longer ones, creating a distinct pattern.
4. Southern Blotting (Optional): In some cases, the DNA fragments are transferred to a membrane using Southern blotting, and a specific probe complementary to the region of interest is used to detect specific DNA fragments.
5. Data Analysis: The banding patterns produced by the digested DNA are analyzed. Differences in the size of fragments between individuals or species reveal the polymorphisms in the DNA sequences, which serve as genetic markers. ⁽¹⁴⁾

CONCLUSION

DNA fingerprinting techniques have revolutionized the identification and authentication of drugs of natural origin, particularly in the fields of herbal medicine and pharmaceutical research. By utilizing the genetic uniqueness of species, these techniques ensure the accurate identification of plant and animal materials used in drug formulations, enhancing quality control, preventing adulteration, and safeguarding consumer safety.

Techniques such as Random Amplified Polymorphic DNA (RAPD), Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeats (SSR), Restriction Fragment Length Polymorphism (RFLP), DNA Barcoding, and Sequence Characterized Amplified Region (SCAR) offer varying levels of specificity, sensitivity, and applicability depending on the complexity of the sample and the information required. RAPD and AFLP are useful for detecting genetic diversity, while DNA barcoding and SCAR provide highly specific markers for species identification. RFLP offers stable co-dominant markers but is more labor-intensive, and SSR is ideal for studying genetic variation within species.

These methods play a critical role in ensuring that natural drugs are derived from the correct species, maintaining the integrity of traditional herbal medicine practices, and meeting modern standards for drug safety and efficacy. They provide essential tools for regulatory bodies, pharmaceutical companies, and researchers in the fight against adulteration, contamination, and substitution in the natural drug industry. As the demand for natural products continues to rise, DNA fingerprinting will remain a cornerstone for verifying the authenticity and purity of these drugs.

In conclusion, DNA fingerprinting techniques have become indispensable in the identification and authentication of natural drugs, promoting the responsible use of biodiversity while ensuring the safety, effectiveness, and authenticity of medicinal products derived from nature.

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