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.
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