Isolation of taq polymerase gene and cloning into e.coli using pgemt vector
Journal name: World Journal of Pharmaceutical Research
Original article title: Isolation of taq polymerase gene & cloning into e.coli using pgemt vector
The WJPR includes peer-reviewed publications such as scientific research papers, reports, review articles, company news, thesis reports and case studies in areas of Biology, Pharmaceutical industries and Chemical technology while incorporating ancient fields of knowledge such combining Ayurveda with scientific data.
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Meenakshi Lal and Sanjeev Ranjan
World Journal of Pharmaceutical Research:
(An ISO 9001:2015 Certified International Journal)
Full text available for: Isolation of taq polymerase gene & cloning into e.coli using pgemt vector
Source type: An International Peer Reviewed Journal for Pharmaceutical and Medical and Scientific Research
Doi: 10.20959/wjpr202010-18536
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Summary of article contents:
Introduction
The article examines the isolation of the Taq polymerase gene and its successful cloning into the bacteria Escherichia coli using the pGEMT vector. Taq polymerase, derived from the thermophilic bacterium Thermus aquaticus, is a thermostable enzyme pivotal for Polymerase Chain Reaction (PCR) applications. Its unique properties not only simplify the reaction process by eliminating the need to add fresh enzyme during thermal cycling but also catalyze DNA amplification efficiently at elevated temperatures. Given its significant role in molecular biology and the substantial global market accompanying its use, there is a growing demand for simplified cloning techniques to produce Taq polymerase in easily manageable host organisms.
Importance of Taq Polymerase
Taq polymerase is integral to PCR, facilitating the amplification of specific DNA sequences. This enzyme functions optimally at high temperatures, with maximal activity observed at around 97.5°C, making it particularly suitable for robust DNA replication. In contrast to other polymerases lacking proofreading ability, the persistence of Taq polymerase in the marketplace can be attributed to its overall robustness, although some alternatives provide additional fidelity through proofreading mechanisms. Its terminal transferase activity, giving rise to A-overhangs during amplification, further positions Taq polymerase as a valuable tool in cloning applications, particularly for TA cloning techniques.
Cloning Strategy
The cloning process outlined in the study involved utilizing a culture of Thermus aquaticus to isolate genomic DNA, from which the Taq polymerase gene was amplified using specifically designed primers. The PCR amplification ensured that the target gene was effectively isolated, confirmed by gel electrophoresis. Subsequently, the amplified fragment was ligated with the pGEMT vector, which is particularly favorable for cloning due to its T-tailed structure. This ligation method enhances the efficiency of incorporating the Taq gene into the plasmid vector before transformation into competent E. coli cells for propagation.
Transformation and Confirmation
Following the ligation process, competent E. coli cells were transformed with the Taq/pGEMT construct through a heat shock mechanism. Transformation was successful, as evidenced by the growth of colonies on selective media containing ampicillin. The isolated plasmid DNA from one of the transformed colonies was analyzed, showing the expected linear, circular, and supercoiled forms that indicate viable clones. Further verification through restriction digestion with the NotI enzyme confirmed the presence of the Taq gene within the pGEMT vector, evidenced by the release of an expected band size on an agarose gel.
Conclusion
The study successfully demonstrated the isolation and cloning of the Taq polymerase gene into the pGEMT vector for introduction into E. coli, providing a streamlined method for producing this essential enzyme. This approach not only facilitates further research on Taq polymerase, including expression studies and potential enhancements of the enzyme’s capabilities through mutagenesis, but also illustrates the efficiency of the cloning technique employed. The advancements in this cloning process are anticipated to support broader applications in molecular biology, particularly in gene therapy and diagnostics, thus solidifying the importance of Taq polymerase as a cornerstone of modern DNA manipulative techniques.
FAQ section (important questions/answers):
What is Taq DNA Polymerase and its significance in research?
Taq DNA Polymerase is a thermostable enzyme from Thermus aquaticus. Its stability at high temperatures allows it to amplify DNA, making it crucial for various molecular biology applications, including PCR, gene cloning, and diagnostics.
How was the Taq polymerase gene isolated in this study?
The Taq polymerase gene was isolated using genomic DNA from Thermus aquaticus. This involved cell lysis with SDS, protein removal with proteinase K, and purification through phenol-chloroform extraction.
Which method was used to amplify the Taq polymerase gene?
The Taq polymerase gene was amplified using PCR with specific primers designed for the gene. The reaction conditions included a series of temperature cycles optimizing DNA denaturation, annealing, and extension.
What vector was used for cloning the Taq polymerase gene?
The pGEMT vector was utilized for cloning the Taq polymerase gene due to its T-tailed ends that facilitate easier ligation of the PCR product, enhancing cloning efficiency.
How was successful transformation of E. coli confirmed?
Transformation success was confirmed by selecting colonies on LB agar plates containing ampicillin. Only E. coli cells containing the plasmid with Taq polymerase could grow in the presence of this antibiotic.
What evidence confirmed the Taq polymerase gene was successfully cloned?
The cloning was confirmed through restriction digestion analysis of plasmid DNA, which generated a specific band size on agarose gel. This indicated the presence of the Taq gene in the pGEMT vector.
Glossary definitions and references:
Scientific and Ayurvedic Glossary list for “Isolation of taq polymerase gene and cloning into e.coli using pgemt vector”. This list explains important keywords that occur in this article and links it to the glossary for a better understanding of that concept in the context of Ayurveda and other topics.
1) Ranjan (Ramjan):
Ranjan refers to one of the authors of the study. Authors play a critical role in establishing credibility and ownership of research work. The name exemplifies the human element in scientific inquiry, illustrating how individual contributions amplify our understanding of complex subjects like molecular biology and cloning techniques. Ranjan's involvement signifies collaboration in achieving meaningful scientific outcomes.
2) Transformation (Transform, Transforming):
Transformed indicates the successful introduction of foreign DNA into a bacterial host, ultimately leading to the expression of desired genes. The term is integral to genetic engineering, as transformation is a pivotal step in cloning and the production of recombinant proteins. It is critical for applications ranging from industrial biotechnology to therapeutic developments.
3) Activity:
Activity signifies the function and effectiveness of an enzyme within a biotechnological process. In the study, Taq polymerase's activity is crucial for PCR applications. High enzyme activity leads to efficient DNA amplification, enabling various applications in molecular biology, including cloning, sequencing, and diagnostics, which are fundamental for research and clinical use.
4) Life:
Life represents the foundation of biological research and pertains directly to living organisms. It encompasses the vital processes that are exploited in experiments, such as cellular diversity, genetics, and metabolism. By understanding the principles of life, researchers can explore the mechanisms governing biological systems, paving the way for innovations in medicine and environmental science.
5) Science (Scientific):
Science serves as the overarching discipline through which the study and exploration of natural phenomena occur. This includes molecular biology, genetics, and biochemistry, all critical to the research presented. The methodology and systematic approach inherent to science provide a framework for deriving valid conclusions and advancing knowledge in diverse fields.
6) Table:
Table refers to a systematic arrangement of data or information, often used to summarize or present experimental results in a clear and organized manner. In scientific writing, tables facilitate comprehension and comparison, enabling readers to quickly assess findings, such as PCR reaction compositions and protocols employed throughout the study.
7) India:
India represents the geographic and institutional context in which the research was conducted. The country hosts a growing scientific community, focusing on biotechnology and molecular biology. Conducting research in India illustrates the significance of global collaboration, showcasing contributions from various nations in addressing scientific challenges and advancing technological frontiers.
8) Purification:
Purification involves techniques employed to isolate and concentrate specific biomolecules, such as nucleic acids or proteins, from complex mixtures. In this study, the purification of genomic DNA is essential for obtaining high-quality samples for PCR amplification. Effective purification is crucial for the reliability of experimental results and the reproducibility of research findings.
9) Performance:
Performance refers to the efficacy and reliability of an enzyme or process in a given context. In genetic engineering, the performance of Taq polymerase relates to its efficiency in amplifying DNA during PCR. Understanding performance metrics enables researchers to select the appropriate enzymes or methods, optimizing outcomes for various applications in biotechnology.
10) Meenakshi:
Meenakshi is another author of the study, contributing to the research efforts alongside Ranjan. The inclusion of authors' names emphasizes collaboration and the collective nature of scientific inquiry. Each author's expertise and role enhance the validity of the research and signal the institutional affiliation that supports scientific investigations.
11) Mutation:
Mutation refers to a change in the nucleotide sequence of DNA, which can impact gene function and protein performance. In molecular biology, understanding mutations is vital for gene manipulation and engineering. This becomes particularly important in developing improved enzyme variants or studying genetic disorders, showcasing the breadth of applications in biomedical research.
12) Heating:
Heating refers to the application of thermal energy during various experimental procedures, notably in PCR and enzyme reactions. Effective heating protocols enable the denaturation of DNA and the optimal functioning of heat-stable enzymes. Mastering heating conditions is essential for successful DNA amplification, influencing experimental outcomes and advancing molecular biology techniques.
13) Water:
Water serves as the primary solvent in biological systems and experiments, crucial for various biochemical processes. In PCR, water is a key component of reaction mixtures, providing the medium necessary for enzyme activity and nucleotide interactions. Its role highlights the importance of environmental conditions in molecular biology experiments and biological reactions.
14) Study (Studying):
Study refers to the systematic investigation conducted to explore and elucidate scientific questions or concepts. The study on the isolation and cloning of the Taq polymerase gene exemplifies the inquiry-driven nature of science. Each study contributes to the broader knowledge base, facilitating advancements in technology, medicine, and our understanding of life.
15) Wall:
Wall refers to the cellular structure that provides support and protection to cells. In the context of the study, breaking down cell walls is critical for isolating genomic DNA and enabling transformation processes. Understanding cell wall composition and structure is essential for advancing techniques in microbiology and molecular biology.
16) Post:
Post refers to the evaluation phase following experimental procedures, where results are analyzed and interpreted. In research, post-experimental analysis serves to validate findings, assess the effectiveness of techniques, and identify areas for further investigation. This crucial step in the research process underlines the iterative nature of scientific inquiry and knowledge development.
Other Science Concepts:
Discover the significance of concepts within the article: ‘Isolation of taq polymerase gene and cloning into e.coli using pgemt vector’. Further sources in the context of Science might help you critically compare this page with similair documents:
Polymerase Chain Reaction (PCR), DNA fingerprinting, DNA Analysis, PCR amplification, Gel electrophoresis, Ethanol precipitation, Gene cloning, Molecular diagnostics.