Lecture 13 - Gene Cloning | Plant Biotechnology - Botany PDF Download

 Page 1


                        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.