Lecture 12 - Cloning Vectors | Plant Biotechnology - Botany PDF Download

 Page 1


                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
Lesson developed under NME-ICT 
Course: Botany 
Subject: Plant Biotechnology 
National Coordinator: Prof. S.C. Bhatla 
Lesson: Cloning Vectors 
Lesson Developer: Dr. Vibha G. Checker 
College / Department: Botany Department, Kirori Mal College, 
University of Delhi 
Reviewer: Prof. Suresh Chand, Professor and Head, School of Life 
Sciences, Devi Ahilya Vishwavidyalaya, Indore. 
 
Language editor: Ms. Manisha Sharma 
Department: Department of Genetics 
Editor: Dr Rama Sisodia 
  
Page 2


                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
Lesson developed under NME-ICT 
Course: Botany 
Subject: Plant Biotechnology 
National Coordinator: Prof. S.C. Bhatla 
Lesson: Cloning Vectors 
Lesson Developer: Dr. Vibha G. Checker 
College / Department: Botany Department, Kirori Mal College, 
University of Delhi 
Reviewer: Prof. Suresh Chand, Professor and Head, School of Life 
Sciences, Devi Ahilya Vishwavidyalaya, Indore. 
 
Language editor: Ms. Manisha Sharma 
Department: Department of Genetics 
Editor: Dr Rama Sisodia 
  
                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
Table of Contents 
Chapter – Cloning Vectors 
• Introduction 
? Plasmids as Cloning Vectors  
? Purpose build plasmid vectors: pBR322 and pUC 
series 
o pBR322 
o pUC series  
? Ti Plasmid 
? Bacterial Artificial Chromosomes (BACs)  
• Bacteriophages as vectors 
? Lambda ( ?) Phage 
o ? Insertion vectors  
o ? Replacement or Substitution vectors 
? M13 Phage 
• Phagemids/ Phasmids 
• Cosmids 
• Shuttle vectors 
• Yeast artificial chromosome 
• P1 artificial chromosome 
• Mouse artificial chromosome 
• Human artificial chromosome 
• Expression vectors 
• Summary 
• Exercise 
• Glossary 
• References 
• Links for animations 
  
Page 3


                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
Lesson developed under NME-ICT 
Course: Botany 
Subject: Plant Biotechnology 
National Coordinator: Prof. S.C. Bhatla 
Lesson: Cloning Vectors 
Lesson Developer: Dr. Vibha G. Checker 
College / Department: Botany Department, Kirori Mal College, 
University of Delhi 
Reviewer: Prof. Suresh Chand, Professor and Head, School of Life 
Sciences, Devi Ahilya Vishwavidyalaya, Indore. 
 
Language editor: Ms. Manisha Sharma 
Department: Department of Genetics 
Editor: Dr Rama Sisodia 
  
                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
Table of Contents 
Chapter – Cloning Vectors 
• Introduction 
? Plasmids as Cloning Vectors  
? Purpose build plasmid vectors: pBR322 and pUC 
series 
o pBR322 
o pUC series  
? Ti Plasmid 
? Bacterial Artificial Chromosomes (BACs)  
• Bacteriophages as vectors 
? Lambda ( ?) Phage 
o ? Insertion vectors  
o ? Replacement or Substitution vectors 
? M13 Phage 
• Phagemids/ Phasmids 
• Cosmids 
• Shuttle vectors 
• Yeast artificial chromosome 
• P1 artificial chromosome 
• Mouse artificial chromosome 
• Human artificial chromosome 
• Expression vectors 
• Summary 
• Exercise 
• Glossary 
• References 
• Links for animations 
  
                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
Introduction 
Gene manipulation has revolutionized research. ‘Vector’in essence refers to a delivery 
agent/vehicle/carrier. Cloning vector is a small molecule of DNA that can carry foreign DNA 
into ahost cell and is capable of self-replication.  
 
 
Source:http://genomicscience.energy.gov.  
Different types of cloning vectors are available, each vector designed to perform a specific 
function.  
Choice of the right cloning vector is an important aspect of cloning. There are certain 
essential properties which a cloning vector must possess: 
Page 4


                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
Lesson developed under NME-ICT 
Course: Botany 
Subject: Plant Biotechnology 
National Coordinator: Prof. S.C. Bhatla 
Lesson: Cloning Vectors 
Lesson Developer: Dr. Vibha G. Checker 
College / Department: Botany Department, Kirori Mal College, 
University of Delhi 
Reviewer: Prof. Suresh Chand, Professor and Head, School of Life 
Sciences, Devi Ahilya Vishwavidyalaya, Indore. 
 
Language editor: Ms. Manisha Sharma 
Department: Department of Genetics 
Editor: Dr Rama Sisodia 
  
                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
Table of Contents 
Chapter – Cloning Vectors 
• Introduction 
? Plasmids as Cloning Vectors  
? Purpose build plasmid vectors: pBR322 and pUC 
series 
o pBR322 
o pUC series  
? Ti Plasmid 
? Bacterial Artificial Chromosomes (BACs)  
• Bacteriophages as vectors 
? Lambda ( ?) Phage 
o ? Insertion vectors  
o ? Replacement or Substitution vectors 
? M13 Phage 
• Phagemids/ Phasmids 
• Cosmids 
• Shuttle vectors 
• Yeast artificial chromosome 
• P1 artificial chromosome 
• Mouse artificial chromosome 
• Human artificial chromosome 
• Expression vectors 
• Summary 
• Exercise 
• Glossary 
• References 
• Links for animations 
  
                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
Introduction 
Gene manipulation has revolutionized research. ‘Vector’in essence refers to a delivery 
agent/vehicle/carrier. Cloning vector is a small molecule of DNA that can carry foreign DNA 
into ahost cell and is capable of self-replication.  
 
 
Source:http://genomicscience.energy.gov.  
Different types of cloning vectors are available, each vector designed to perform a specific 
function.  
Choice of the right cloning vector is an important aspect of cloning. There are certain 
essential properties which a cloning vector must possess: 
                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
1. It should be able to replicate itself and the DNA segment it carries independently.  
2. It should be of small size, so that it is easy to manipulate. 
3. It should be easy to recover from the host cell. 
4. It should be easily transferred from one cell to another by simple methods. 
5. It should contain several unique restriction enzyme sites. These sites are present 
only once and clustered in one locationand are known as multiple cloning sites or 
polylinker. Restriction enzyme site is cleaved with a restriction enzyme to open the 
cloning vector without disrupting any other region. Insert DNA fragment is digested 
with the same enzyme and ligated with the vector. 
6. There should be easy ways to detect their presence in host organisms. Bacterial 
cloning plasmids carry an antibiotic resistance gene to distinguish the host cells that 
carry vectors from the host cells that do not contain vectors. 
7. There should be easy ways to detect the presence of inserted DNA. 
 
The first recombinant DNA 
 
 
 
 
 
 
 
 
 
 
 
Video:Stanley Cohen and Herbert Boyer's historic experiment used techniques to cut and 
paste DNA to create the first custom-made organism containing recombined or 
"recombinant" DNA. 
Source:http://www.dnalc.org/view/15915-The-first-recombinant-DNA.html 
 
 
Page 5


                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
 
 
 
 
 
 
 
Lesson developed under NME-ICT 
Course: Botany 
Subject: Plant Biotechnology 
National Coordinator: Prof. S.C. Bhatla 
Lesson: Cloning Vectors 
Lesson Developer: Dr. Vibha G. Checker 
College / Department: Botany Department, Kirori Mal College, 
University of Delhi 
Reviewer: Prof. Suresh Chand, Professor and Head, School of Life 
Sciences, Devi Ahilya Vishwavidyalaya, Indore. 
 
Language editor: Ms. Manisha Sharma 
Department: Department of Genetics 
Editor: Dr Rama Sisodia 
  
                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
Table of Contents 
Chapter – Cloning Vectors 
• Introduction 
? Plasmids as Cloning Vectors  
? Purpose build plasmid vectors: pBR322 and pUC 
series 
o pBR322 
o pUC series  
? Ti Plasmid 
? Bacterial Artificial Chromosomes (BACs)  
• Bacteriophages as vectors 
? Lambda ( ?) Phage 
o ? Insertion vectors  
o ? Replacement or Substitution vectors 
? M13 Phage 
• Phagemids/ Phasmids 
• Cosmids 
• Shuttle vectors 
• Yeast artificial chromosome 
• P1 artificial chromosome 
• Mouse artificial chromosome 
• Human artificial chromosome 
• Expression vectors 
• Summary 
• Exercise 
• Glossary 
• References 
• Links for animations 
  
                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
Introduction 
Gene manipulation has revolutionized research. ‘Vector’in essence refers to a delivery 
agent/vehicle/carrier. Cloning vector is a small molecule of DNA that can carry foreign DNA 
into ahost cell and is capable of self-replication.  
 
 
Source:http://genomicscience.energy.gov.  
Different types of cloning vectors are available, each vector designed to perform a specific 
function.  
Choice of the right cloning vector is an important aspect of cloning. There are certain 
essential properties which a cloning vector must possess: 
                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
1. It should be able to replicate itself and the DNA segment it carries independently.  
2. It should be of small size, so that it is easy to manipulate. 
3. It should be easy to recover from the host cell. 
4. It should be easily transferred from one cell to another by simple methods. 
5. It should contain several unique restriction enzyme sites. These sites are present 
only once and clustered in one locationand are known as multiple cloning sites or 
polylinker. Restriction enzyme site is cleaved with a restriction enzyme to open the 
cloning vector without disrupting any other region. Insert DNA fragment is digested 
with the same enzyme and ligated with the vector. 
6. There should be easy ways to detect their presence in host organisms. Bacterial 
cloning plasmids carry an antibiotic resistance gene to distinguish the host cells that 
carry vectors from the host cells that do not contain vectors. 
7. There should be easy ways to detect the presence of inserted DNA. 
 
The first recombinant DNA 
 
 
 
 
 
 
 
 
 
 
 
Video:Stanley Cohen and Herbert Boyer's historic experiment used techniques to cut and 
paste DNA to create the first custom-made organism containing recombined or 
"recombinant" DNA. 
Source:http://www.dnalc.org/view/15915-The-first-recombinant-DNA.html 
 
 
                     Cloning Vectors 
 
Institute of Lifelong Learning, University of Delhi 
 
 
 
Plasmids as Cloning Vectors  
 
 
 
Animation: Plasmids as cloning vector 
Source:http://www.dnalc.org/view/15476-Mechanism-of-Recombination-3D-
animation-with-with-basic-narration.html 
 
Source:http://upload.wikimedia.org/wikipedia/commons/thumb/c/cf/Plasmid_%28english%
29.svg/1280px-Plasmid_%28english%29.svg.png