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                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
Lesson Prepared Under MHRD project “National Mission on 
Education Through ICT” 
Discipline: Botany 
Paper: Plant Biotechnology 
Lesson: Gene Cloning 
 
Lesson Developer: Dr Neetu Chaudhary
1
, Dr. Parul Agarwal
2
 
College: 
1
Acharya NarenderDev College,
2
Department of Genetics, 
University of Delhi, South Campus 
 
Lesson Reviewer: Prof. Suresh Chand, Professor and Head, School of 
Life Sciences, Devi AhilyaVishwavidyalaya, Indore 
 
Language editor: Ms. Manisha Sharma 
Department: Department of Genetics 
Editor: Dr Rama Sisodia, Fellow in Botany ILLL  
Page 2


                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
Lesson Prepared Under MHRD project “National Mission on 
Education Through ICT” 
Discipline: Botany 
Paper: Plant Biotechnology 
Lesson: Gene Cloning 
 
Lesson Developer: Dr Neetu Chaudhary
1
, Dr. Parul Agarwal
2
 
College: 
1
Acharya NarenderDev College,
2
Department of Genetics, 
University of Delhi, South Campus 
 
Lesson Reviewer: Prof. Suresh Chand, Professor and Head, School of 
Life Sciences, Devi AhilyaVishwavidyalaya, Indore 
 
Language editor: Ms. Manisha Sharma 
Department: Department of Genetics 
Editor: Dr Rama Sisodia, Fellow in Botany ILLL  
                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
Table of Contents 
Chapter: Gene Cloning 
• Introduction 
• Steps in molecular cloning 
• Formation of recombinant DNA molecule 
? Choice of host organism and cloning vector 
? Preparation of DNA to be cloned 
? Preparation of vector DNA 
? Creation of recombinant DNA with DNA ligase 
• Introduction of recombinant DNA into host organism 
• Selection of organisms containing vector sequences 
• Screening for clones having desired DNA inserts and biological 
properties 
• PCR-mediated gene cloning 
• TA cloning 
• Blunt-end cloning 
• cDNA cloning 
• Long distance PCR 
• Hot start PCR 
• Touchdown PCR 
• Gene construct 
• Expression systems 
• Summary 
• Exercise/practice 
• Glossary 
Page 3


                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
Lesson Prepared Under MHRD project “National Mission on 
Education Through ICT” 
Discipline: Botany 
Paper: Plant Biotechnology 
Lesson: Gene Cloning 
 
Lesson Developer: Dr Neetu Chaudhary
1
, Dr. Parul Agarwal
2
 
College: 
1
Acharya NarenderDev College,
2
Department of Genetics, 
University of Delhi, South Campus 
 
Lesson Reviewer: Prof. Suresh Chand, Professor and Head, School of 
Life Sciences, Devi AhilyaVishwavidyalaya, Indore 
 
Language editor: Ms. Manisha Sharma 
Department: Department of Genetics 
Editor: Dr Rama Sisodia, Fellow in Botany ILLL  
                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
Table of Contents 
Chapter: Gene Cloning 
• Introduction 
• Steps in molecular cloning 
• Formation of recombinant DNA molecule 
? Choice of host organism and cloning vector 
? Preparation of DNA to be cloned 
? Preparation of vector DNA 
? Creation of recombinant DNA with DNA ligase 
• Introduction of recombinant DNA into host organism 
• Selection of organisms containing vector sequences 
• Screening for clones having desired DNA inserts and biological 
properties 
• PCR-mediated gene cloning 
• TA cloning 
• Blunt-end cloning 
• cDNA cloning 
• Long distance PCR 
• Hot start PCR 
• Touchdown PCR 
• Gene construct 
• Expression systems 
• Summary 
• Exercise/practice 
• Glossary 
                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
• References/Bibliography/Further Reading 
• Web links  
Page 4


                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
Lesson Prepared Under MHRD project “National Mission on 
Education Through ICT” 
Discipline: Botany 
Paper: Plant Biotechnology 
Lesson: Gene Cloning 
 
Lesson Developer: Dr Neetu Chaudhary
1
, Dr. Parul Agarwal
2
 
College: 
1
Acharya NarenderDev College,
2
Department of Genetics, 
University of Delhi, South Campus 
 
Lesson Reviewer: Prof. Suresh Chand, Professor and Head, School of 
Life Sciences, Devi AhilyaVishwavidyalaya, Indore 
 
Language editor: Ms. Manisha Sharma 
Department: Department of Genetics 
Editor: Dr Rama Sisodia, Fellow in Botany ILLL  
                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
Table of Contents 
Chapter: Gene Cloning 
• Introduction 
• Steps in molecular cloning 
• Formation of recombinant DNA molecule 
? Choice of host organism and cloning vector 
? Preparation of DNA to be cloned 
? Preparation of vector DNA 
? Creation of recombinant DNA with DNA ligase 
• Introduction of recombinant DNA into host organism 
• Selection of organisms containing vector sequences 
• Screening for clones having desired DNA inserts and biological 
properties 
• PCR-mediated gene cloning 
• TA cloning 
• Blunt-end cloning 
• cDNA cloning 
• Long distance PCR 
• Hot start PCR 
• Touchdown PCR 
• Gene construct 
• Expression systems 
• Summary 
• Exercise/practice 
• Glossary 
                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
• References/Bibliography/Further Reading 
• Web links  
                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
Introduction 
The word cloning means creating a perfect replica or genetically identical copy. In nature, 
genetically identical organismsor ‘clones’ are created by the process of asexual 
reproduction.The technique of generating identical copies of short stretches of DNA is 
referred to as molecular cloning. 
DNA cloning refers to replication of a single DNA molecule to create a large number of 
identical DNA molecules. Molecular cloning experiments to create recombinant DNA 
generally use DNA sequences from two different organisms: one as the source of DNA to be 
cloned, and other is the cloning vector required for replication of the recombinant DNA 
inside the host cell. These cloning experiments are based on the fact that DNA structure is 
fundamentally same in all living organisms. A region of DNA (gene) from one organism can 
be easily inserted into another organism’sDNA segment due to the same chemical structure. 
This recombinant DNA should haveregulatory sequences required forreplication when 
introduced into a host organism. In bacteria or yeast, where the recombinant DNA is 
inserted in the form of a plasmid, the plasmid replicates independently of the host 
organism’s chromosome, while in organisms where recombinant DNA is inserted into the 
chromosome itself, replication of the recombinant DNA occurs along with the host cell’s 
chromosomal replication. 
 
 
Page 5


                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
Lesson Prepared Under MHRD project “National Mission on 
Education Through ICT” 
Discipline: Botany 
Paper: Plant Biotechnology 
Lesson: Gene Cloning 
 
Lesson Developer: Dr Neetu Chaudhary
1
, Dr. Parul Agarwal
2
 
College: 
1
Acharya NarenderDev College,
2
Department of Genetics, 
University of Delhi, South Campus 
 
Lesson Reviewer: Prof. Suresh Chand, Professor and Head, School of 
Life Sciences, Devi AhilyaVishwavidyalaya, Indore 
 
Language editor: Ms. Manisha Sharma 
Department: Department of Genetics 
Editor: Dr Rama Sisodia, Fellow in Botany ILLL  
                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
Table of Contents 
Chapter: Gene Cloning 
• Introduction 
• Steps in molecular cloning 
• Formation of recombinant DNA molecule 
? Choice of host organism and cloning vector 
? Preparation of DNA to be cloned 
? Preparation of vector DNA 
? Creation of recombinant DNA with DNA ligase 
• Introduction of recombinant DNA into host organism 
• Selection of organisms containing vector sequences 
• Screening for clones having desired DNA inserts and biological 
properties 
• PCR-mediated gene cloning 
• TA cloning 
• Blunt-end cloning 
• cDNA cloning 
• Long distance PCR 
• Hot start PCR 
• Touchdown PCR 
• Gene construct 
• Expression systems 
• Summary 
• Exercise/practice 
• Glossary 
                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
• References/Bibliography/Further Reading 
• Web links  
                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
Introduction 
The word cloning means creating a perfect replica or genetically identical copy. In nature, 
genetically identical organismsor ‘clones’ are created by the process of asexual 
reproduction.The technique of generating identical copies of short stretches of DNA is 
referred to as molecular cloning. 
DNA cloning refers to replication of a single DNA molecule to create a large number of 
identical DNA molecules. Molecular cloning experiments to create recombinant DNA 
generally use DNA sequences from two different organisms: one as the source of DNA to be 
cloned, and other is the cloning vector required for replication of the recombinant DNA 
inside the host cell. These cloning experiments are based on the fact that DNA structure is 
fundamentally same in all living organisms. A region of DNA (gene) from one organism can 
be easily inserted into another organism’sDNA segment due to the same chemical structure. 
This recombinant DNA should haveregulatory sequences required forreplication when 
introduced into a host organism. In bacteria or yeast, where the recombinant DNA is 
inserted in the form of a plasmid, the plasmid replicates independently of the host 
organism’s chromosome, while in organisms where recombinant DNA is inserted into the 
chromosome itself, replication of the recombinant DNA occurs along with the host cell’s 
chromosomal replication. 
 
 
                        Gene Cloning  
 
Institute of Lifelong Learning, University of Delhi 
 
A 
 
 
B 
Figure:Diagrammatic representation of gene cloning (A) Principle of gene cloning (B) 
Methodology followed for gene cloning. 
Source: http://www.biofortified.org/2014/03/e-coli-in-gmos/ (cc), 
http://hubpages.com/hub/Molecular-Biology-Laboratory-Techniques#slide6656310 
 
Gene cloning is quite similar to PCR asboth of these techniquesresultingeneration of multiple 
copies of DNA sequence. However,gene cloning replicates DNA in a living host organism, 
while PCR replicates DNA without using living cells. 
 
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23 docs

FAQs on Lecture 13 - Gene Cloning - Plant Biotechnology - Botany

1. What is gene cloning in botany?
Gene cloning in botany refers to the process of isolating and making copies of a specific gene or DNA segment from one organism and inserting it into another organism. This technique allows scientists to study and manipulate genes, understand their functions, and potentially introduce desirable traits into plants for agricultural purposes.
2. How is gene cloning performed in botany?
Gene cloning in botany involves several steps. First, the target gene of interest is isolated from the source organism using enzymes that cut DNA at specific sites. Next, a plasmid (a small, circular DNA molecule) is extracted from bacteria and cut with the same enzymes. The target gene is then inserted into the cut plasmid using DNA ligase, creating a recombinant DNA molecule. This recombinant DNA is then introduced into host plant cells using various methods such as Agrobacterium-mediated transformation or gene gun bombardment. Finally, the transformed plant cells are cultured and grown into whole plants, which carry the cloned gene.
3. What are the applications of gene cloning in botany?
Gene cloning has numerous applications in botany. It allows scientists to study gene functions by manipulating their expression levels or introducing mutations. It also enables the production of genetically modified plants with desirable traits such as increased resistance to pests, diseases, or environmental stresses. Gene cloning is used in plant breeding to introduce specific traits into crop plants, improving their yield, nutritional content, or tolerance to harsh conditions. Additionally, it plays a crucial role in pharmaceutical production, as plants can be engineered to produce valuable proteins or secondary metabolites.
4. What are the challenges in gene cloning in botany?
Gene cloning in botany faces several challenges. One major challenge is the difficulty in introducing foreign genes into plant cells and achieving stable integration into the plant genome. Plants have complex cell walls and efficient defense mechanisms that make genetic transformation challenging. Another challenge is ensuring proper gene expression and regulation in the transformed plants. Gene silencing or unintended effects on other plant genes can occur, affecting the desired trait or causing unintended consequences. Additionally, ethical and environmental concerns regarding genetically modified plants pose challenges in the acceptance and implementation of gene cloning technologies.
5. What are the potential benefits of gene cloning in botany?
Gene cloning in botany offers various potential benefits. It allows for the development of genetically modified plants with improved traits, such as increased yield, enhanced nutritional value, or resistance to pests and diseases. This can contribute to food security, reduce pesticide use, and improve agricultural sustainability. Gene cloning also enables the production of valuable pharmaceutical compounds in plants, offering a cost-effective and scalable method for pharmaceutical production. Furthermore, it provides a deeper understanding of plant genetics and gene functions, aiding in the development of new plant varieties and the conservation of endangered plant species.
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