Page 1
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
Lesson Prepared Under MHRD project “ National Mission on
Education Through ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: Enzymes used in Recombinant DNA Technology
Lesson Developer:DR. Priyanka Deveshwar
Department/College: Gargi College, University of Delhi
Lesson Reviewer:Dr Parul Agarwal
Department/College:Department of Genetics, University of Delhi
South Campus
Language Editor:Manisha Sharma
Department: Department of Genetics, University of Delhi South
Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Page 2
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
Lesson Prepared Under MHRD project “ National Mission on
Education Through ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: Enzymes used in Recombinant DNA Technology
Lesson Developer:DR. Priyanka Deveshwar
Department/College: Gargi College, University of Delhi
Lesson Reviewer:Dr Parul Agarwal
Department/College:Department of Genetics, University of Delhi
South Campus
Language Editor:Manisha Sharma
Department: Department of Genetics, University of Delhi South
Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
Table of Contents
Chapter: Enzymes used in Recombinant DNA Technology
• Introduction
• Variety of DNA modifying enzymes
• Nucleases
• Ligases
• Polymerases
• DNA modifying enzymes
• Alkaline Phosphatase
• Polynucleotide Kinase
• Terminal Transferase
• Molecular Scissors: Restriction Endonucleases
• Discovery and Biological Function of Restriction
Endonucleases
• Types of restriction endonucleases
• Nomenclature of restriction endonucleases
• Features of recognition sequences and cleavage sites
of type II restriction endonucleases
• Symmetry
• Length and Frequency
• Blunt Ends and Sticky Ends
• Isoschizomers
• Restriction Mapping
• Applications of Restriction Enzymes
• Summary
• Exercise
• Glossary
Page 3
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
Lesson Prepared Under MHRD project “ National Mission on
Education Through ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: Enzymes used in Recombinant DNA Technology
Lesson Developer:DR. Priyanka Deveshwar
Department/College: Gargi College, University of Delhi
Lesson Reviewer:Dr Parul Agarwal
Department/College:Department of Genetics, University of Delhi
South Campus
Language Editor:Manisha Sharma
Department: Department of Genetics, University of Delhi South
Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
Table of Contents
Chapter: Enzymes used in Recombinant DNA Technology
• Introduction
• Variety of DNA modifying enzymes
• Nucleases
• Ligases
• Polymerases
• DNA modifying enzymes
• Alkaline Phosphatase
• Polynucleotide Kinase
• Terminal Transferase
• Molecular Scissors: Restriction Endonucleases
• Discovery and Biological Function of Restriction
Endonucleases
• Types of restriction endonucleases
• Nomenclature of restriction endonucleases
• Features of recognition sequences and cleavage sites
of type II restriction endonucleases
• Symmetry
• Length and Frequency
• Blunt Ends and Sticky Ends
• Isoschizomers
• Restriction Mapping
• Applications of Restriction Enzymes
• Summary
• Exercise
• Glossary
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
• References
Page 4
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
Lesson Prepared Under MHRD project “ National Mission on
Education Through ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: Enzymes used in Recombinant DNA Technology
Lesson Developer:DR. Priyanka Deveshwar
Department/College: Gargi College, University of Delhi
Lesson Reviewer:Dr Parul Agarwal
Department/College:Department of Genetics, University of Delhi
South Campus
Language Editor:Manisha Sharma
Department: Department of Genetics, University of Delhi South
Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
Table of Contents
Chapter: Enzymes used in Recombinant DNA Technology
• Introduction
• Variety of DNA modifying enzymes
• Nucleases
• Ligases
• Polymerases
• DNA modifying enzymes
• Alkaline Phosphatase
• Polynucleotide Kinase
• Terminal Transferase
• Molecular Scissors: Restriction Endonucleases
• Discovery and Biological Function of Restriction
Endonucleases
• Types of restriction endonucleases
• Nomenclature of restriction endonucleases
• Features of recognition sequences and cleavage sites
of type II restriction endonucleases
• Symmetry
• Length and Frequency
• Blunt Ends and Sticky Ends
• Isoschizomers
• Restriction Mapping
• Applications of Restriction Enzymes
• Summary
• Exercise
• Glossary
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
• References
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
Introduction
Recombinant DNA technology includes the procedures forcreating recombinant DNA
(rDNA). rDNA is a recombinant molecule where the vector is joined with a natural or
asynthetic DNA segment of interest to make a moleculethat can replicate in a living
cell.To produce rDNA one must be able to cut the vector at precise sites so that the DNA
of interest can be inserted. The digested vector molecule and the DNA of interest are
joined together to form a rDNA molecule. In the last few years, many new technologies
for the manipulation of DNA have been developed. These techniques allow not only
cutting and joining of DNA, but also shortening, lengthening and amplification of DNA
molecules, copying into RNA and also development of new and modified DNA molecules
by the addition or removal of specific chemical groups.These manipulations have not
only led to genetic engineering but also increased our knowledge about gene structure
and control of gene expression.
Animportantcomponent of making rDNA is that most of the DNA manipulationsare done
in vitroi.e., outside the living cells. This requires simulation of conditions and apparatus
present in a living cell, in a test tube. In a living cell, enzymes participate in all crucial
processes involved in DNA modifications. These processes includesDNA replication,
transcription, repair ofmutated and damaged DNA, recombination between different DNA
molecules and breakdown of unwanted or foreign DNA (for example invading viral DNA).
If these enzymes can be isolated and purified, the above said processes can be done in a
test tube (artificial conditions), provided that all catalytic requirements of the enzymes
are fulfilled. Purified and high quality DNA modifying enzymes holds the key to the
essence of genetic engineering. This has resulted in development of a big industry
involved in production and supply of purified DNA modifying enzymes targeting the
molecular biologists.
In this chapter, we will try to have a glance at the variety of DNAmodifying enzymes
available to the molecular biologists. Special emphasis will be given to enzyme
responsible for precise cutting of the DNA molecules called as ‘restriction endonuclease’.
Variety of DNA Modifying Enzymes
DNA recombinant technology requires a whole toolkit for modifying/manipulating DNA.
These tools invariably include a variety of enzymes comprehensively called as DNA
modifying enzymes. These include –
Page 5
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
Lesson Prepared Under MHRD project “ National Mission on
Education Through ICT”
Discipline: Botany
Paper: Plant Biotechnology
National Coordinator: Prof. S.C. Bhatla
Lesson: Enzymes used in Recombinant DNA Technology
Lesson Developer:DR. Priyanka Deveshwar
Department/College: Gargi College, University of Delhi
Lesson Reviewer:Dr Parul Agarwal
Department/College:Department of Genetics, University of Delhi
South Campus
Language Editor:Manisha Sharma
Department: Department of Genetics, University of Delhi South
Campus
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
Table of Contents
Chapter: Enzymes used in Recombinant DNA Technology
• Introduction
• Variety of DNA modifying enzymes
• Nucleases
• Ligases
• Polymerases
• DNA modifying enzymes
• Alkaline Phosphatase
• Polynucleotide Kinase
• Terminal Transferase
• Molecular Scissors: Restriction Endonucleases
• Discovery and Biological Function of Restriction
Endonucleases
• Types of restriction endonucleases
• Nomenclature of restriction endonucleases
• Features of recognition sequences and cleavage sites
of type II restriction endonucleases
• Symmetry
• Length and Frequency
• Blunt Ends and Sticky Ends
• Isoschizomers
• Restriction Mapping
• Applications of Restriction Enzymes
• Summary
• Exercise
• Glossary
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
• References
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
Introduction
Recombinant DNA technology includes the procedures forcreating recombinant DNA
(rDNA). rDNA is a recombinant molecule where the vector is joined with a natural or
asynthetic DNA segment of interest to make a moleculethat can replicate in a living
cell.To produce rDNA one must be able to cut the vector at precise sites so that the DNA
of interest can be inserted. The digested vector molecule and the DNA of interest are
joined together to form a rDNA molecule. In the last few years, many new technologies
for the manipulation of DNA have been developed. These techniques allow not only
cutting and joining of DNA, but also shortening, lengthening and amplification of DNA
molecules, copying into RNA and also development of new and modified DNA molecules
by the addition or removal of specific chemical groups.These manipulations have not
only led to genetic engineering but also increased our knowledge about gene structure
and control of gene expression.
Animportantcomponent of making rDNA is that most of the DNA manipulationsare done
in vitroi.e., outside the living cells. This requires simulation of conditions and apparatus
present in a living cell, in a test tube. In a living cell, enzymes participate in all crucial
processes involved in DNA modifications. These processes includesDNA replication,
transcription, repair ofmutated and damaged DNA, recombination between different DNA
molecules and breakdown of unwanted or foreign DNA (for example invading viral DNA).
If these enzymes can be isolated and purified, the above said processes can be done in a
test tube (artificial conditions), provided that all catalytic requirements of the enzymes
are fulfilled. Purified and high quality DNA modifying enzymes holds the key to the
essence of genetic engineering. This has resulted in development of a big industry
involved in production and supply of purified DNA modifying enzymes targeting the
molecular biologists.
In this chapter, we will try to have a glance at the variety of DNAmodifying enzymes
available to the molecular biologists. Special emphasis will be given to enzyme
responsible for precise cutting of the DNA molecules called as ‘restriction endonuclease’.
Variety of DNA Modifying Enzymes
DNA recombinant technology requires a whole toolkit for modifying/manipulating DNA.
These tools invariably include a variety of enzymes comprehensively called as DNA
modifying enzymes. These include –
Enzymes used in Recombinant DNA
Technology
Institute of Lifelong Learning, University of Delhi
1. Nucleases
2. Ligases
3. Polymerases
4. DNA modifying enzymes
Most of the reactions performed by the abovesaid enzymes cannot be accomplished by
non-enzymatic chemical methods, hence underlining their importance in molecular
cloning.The above said classes of enzymes differ in the reactions they catalyse, but some
enzymes may perform more than one reaction. Also, the enzymes mentioned here are
useful only for DNA manipulation but other similar enzymes modifying RNA are also
available.
Nucleases
Nucleases are enzymes that degrade DNA molecules by breaking the phosphodiester
bonds that link one nucleotide to the next in a DNA strand. Nucleases can be broadly
categorized into
(i) exonucleases and (ii) endonucleases.
Exonuclease removes the terminal nucleotide of the DNA molecule by breaking the
phosphodiester bond, whereas endonuclease breaks the internal phosphodiester bond.
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