Molecular markers are important tools in research, offering a means to identify and measure specific genetic loci within populations. DNA barcoding is a valuable tool for non-expert taxonomists, enhancing the efficiency and accuracy of species identification. These markers, associated with particular genes or traits, facilitate the study of genetic relationships, variety identification, phylogenetic analysis, and gene mapping, including quantitative trait loci (QTLs). They are broadly categorized into two types: hybridization-based and PCR-based markers. Restriction Fragment Length Polymorphism (RFLP) is one of the earliest hybridization-based techniques, detecting DNA sequence variations at restriction enzyme sites. The method involves electrophoresis of DNA fragments, which are then transferred to a membrane and probed with labeled DNA to visualize genetic differences. To enhance specificity and sensitivity, nested PCR can be employed, which involves two rounds of PCR amplification with external and nested primers. PCR-RFLP, an extension of this method, is used for detecting genetic variations such as microindels, single-nucleotide polymorphisms (SNPs), and multi-nucleotide polymorphisms (MNPs). These techniques have been crucial in advancing molecular plant breeding, genomic selection, and genome editing, thereby enriching our understanding of genetic diversity in crops and other organisms

RFLP, PCR, AFLP, Barcoding, Aquaculture

Molecular marking techniques have fundamentally transformed genetic research and applications across diverse fields. These methods facilitate precise identification and measurement of genetic loci, enabling detailed studies of genetic relationships, variety identification, phylogenetic analysis, and gene mapping, including quantitative trait loci (QTLs). The evolution of molecular markers from hybridization-based techniques like Restriction Fragment Length Polymorphism (RFLP) to advanced PCR-based methods and beyond illustrates the dynamic advancements in this field. Hybridization-based markers such as RFLP have laid the groundwork for detecting genetic variations through restriction enzyme sites, while PCR-based markers, including Amplified Fragment Length Polymorphism (AFLP) and Random Amplified Polymorphic DNA (RAPD), offer sensitivity and cost-effectiveness for analyzing genetic diversity. Although techniques like RAPD are valuable for rapid analysis and mapping population genetics, they are often complemented by more precise methods in current research. The development of Genotyping-by-Sequencing (GBS) and Loop-Mediated Isothermal Amplification (LAMP) highlights the shift towards high-throughput and efficient approaches. GBS leverages next-generation sequencing to provide comprehensive genetic insights, while LAMP offers a rapid, user-friendly alternative for gene amplification without the need for complex equipment. These advancements have not only enhanced our understanding of genetic diversity in crops and other organisms but have also significantly contributed to practical applications in areas such as plant breeding, aquaculture, and species identification. By integrating these molecular marking techniques, researchers can achieve more accurate, efficient, and detailed genetic analyses, fostering advancements in both scientific research and applied genetic technologies

Gowhar Iqbal, Shahid Gul, Arya Singh and Ad Viral Kumar Ganpatbhai (2024). Advancements in Molecular Markers: From RFLP to Modern PCR and DNA Barcoding Techniques. International Journal on Emerging Technologies, 15(2): 29–32