28, February 2025

Exploring the Fundamentals of Molecular Biology: From DNA Extraction to Gene Cloning and Biochemical Analysis

Author(s): 1 Gajanan Gite and 2 Aman Tiple

Authors Affiliations:

1,2 Institute of Biosciences and Technology, MGM University, Chh. Sambhajinagar,  Maharashtra, India,

DOIs:10.2015/IJIRMF/202502023     |     Paper ID: IJIRMF202502023


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Molecular biology is essential for understanding biological systems, with DNA extraction, polymerase chain reaction (PCR) analysis, gel electrophoresis, and microbial biochemical testing serving as key methodologies. This study explores the extraction of DNA from bacterial, fungal, and plant samples, highlighting challenges and optimized protocols for high-quality nucleic acid retrieval. PCR analysis is examined in genetic analysis, disease detection, and molecular cloning, emphasizing primer specificity and real-time PCR advancements. Gel electrophoresis is discussed as a crucial tool for verifying PCR products and genetic modifications. Microbiological techniques such as Gram staining and biochemical assays aid in bacterial identification, particularly in sugarcane tissues and fish gut microbiota, providing insights into microbial interactions. Gene cloning methodologies, including restriction enzyme digestion, ligation, and PCR-based cloning, are explored for their applications in biotechnology and medical research. Despite advancements, challenges persist in nucleic acid purification and amplification efficiency. This study summarizes fundamental molecular biology techniques, their applications, and ongoing improvements, offering insights into future research directions.

Molecular biology, DNA extraction, PCR analysis, gel electrophoresis, gene cloning, bacterial identification.

Gajanan Gite and  Aman Tiple (2025); Exploring the Fundamentals of Molecular Biology: From DNA Extraction to Gene Cloning and Biochemical Analysis, International Journal for Innovative Research in Multidisciplinary Field, ISSN(O): 2455-0620, Vol-11, Issue-2, Pp.146-154.          Available on –   https://www.ijirmf.com/

  1. Xu et al., “A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis,” PLoS ONE, vol. 7, no. 1, p. e22900, Jan. 2012, doi: 10.1371/journal.pone.0022900.
  2. Bolano et al., “Rapid methods to extract DNA and RNA fromCryptococcus neoformans,” FEMS Yeast Research, vol. 1, no. 3, pp. 221–224, Dec. 2001, doi: 10.1111/j.1567-1364.2001.tb00037.x.
  3. A. Couch and P. J. Fritz, “Isolation of DNA from plants high in polyphenolics,” Plant Mol. Biol. Rep., vol. 8, no. 1, pp. 8–12, Feb. 1990, doi: 10.1007/bf02668875.
  4. O. Kotchoni, E. W. Gachomo, and J. C. Jimenez-Lopez, “A plant cocktail amenable for PCR-based genetic analysis in Arabidopsis thaliana,” Mol. Biol. Rep., vol. 38, no. 8, pp. 5281–5284, Jan. 2011, doi: 10.1007/s11033-011-0677-6.
  5. Edwards, C. Johnstone, and C. Thompson, “A simple and rapid method for the preparation of plant genomic DNA for PCR analysis,” Nucl Acids Res, vol. 19, no. 6, p. 1349, Jan. 1991, doi: 10.1093/nar/19.6.1349.
  6. Hoseini and M. G. Sauer, “Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering,” J. Biol. Eng., vol. 9, no. 1, p. 2, Jan. 2015, doi: 10.1186/1754-1611-9-2.
  7. Alaey, R. Naderi, A. Vezvaei, A. Khalighi, and A. Salami, “Mitra Aelaei Comparing study between four different methods of genomic DNA extraction from Cyclamen persicum Mill,” Int. J. Agric. Biol., vol. 7, no. 6, pp. 882–884, 2005.
  8. Promega Corporation, Wizard Genomic DNA Purification Kit, Revised 12/14. 2014. [Online]. Available: https://www.promega.com
  9. H. Rêgo et al., “Protocol for Papillomavirus DNA extraction from cervical brushes using the Wizard® Genomic DNA Purification Kit v1,” protocol.io, Mar. 28, 2023. https://doi.org/10.17504/protocols.io.8epv5jp94l1b/v1
  10. OPS Diagnostics, “Protocol for Extracting DNA from Plant Samples Using CTAB.” https://opsdiagnostics.com/notes/protocols/ctab_protocol_for_plants.htm
  11. S. Toni et al., “Optimization of phenol-chloroform RNA extraction,” MethodsX, vol. 5, pp. 599–608, Jan. 2018, doi: 10.1016/j.mex.2018.05.011.
  12. N. Fredricks, C. Smith, and A. Meier, “Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR,” J. Clin. Microbiol., vol. 43, no. 10, pp. 5122–5128, Oct. 2005, doi: 10.1128/jcm.43.10.5122-5128.2005.
  13. Y. Eileen and W. W. Khine, “Fecal Bacterial DNA Extraction V1,” protocols.io, Feb. 18, 2018. https://doi.org/10.17504/protocols.io.nbqdamw
  14. Promega Corporation, “QuantiFluor® dsDNA System,” Technical Manual, 2024. [Online]. Available: https://www.promega.de/-/media/files/resources/protocols/technical-manuals/101/quantifluor-dsdna-system-protocol-tm346.pdf?rev=e4a9ac60a77448108b16293db41935d8&sc_lang=en
  15. Sultana, Md. S. S. Sawrav, Md. B. Rahma, Md. S. Hossain, and Md. A. Haque, “Isolation and Biochemical Characterization of Xylanase Enzyme Producing Bacteria from Goat Rumen,” AIJR Proceedings, pp. 1–8, Feb. 2022, doi: 10.21467/proceedings.123.1.
  16. Samanta, J. Banerjee, K. M. Ali, B. Giri, and S. K. Dash, “Biochemical Characterization and Antibiotic Sensitivity Profiling of Uropathogenic Bacterial Strains Isolated from Urine Sample of Uti Patients,” J. Adv. Sci. Res., vol. 11, no. 1, pp. 280–295, Mar. 2020, [Online]. Available: https://sciensage.info/index.php/JASR/article/view/444
  17. “Cloning – Lecture notes – Product Code: HTBM Number of experiments that can be performed: 5 Duration – Studocu,” Studocu. https://www.studocu.com/in/document/mahatma-gandhi-university/environmental-biotechnology/cloning-lecture-notes/44608230
  18. R. Tuttle, N. D. Trahan, and M. S. Son, “Growth and Maintenance of Escherichia coli Laboratory Strains,” Current Protocols, vol. 1, no. 1, Jan. 2021, doi: 10.1002/cpz1.20.

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