Lecture 11 - Enzymes used in Recombinant DNA Technology | Plant Biotechnology - Botany PDF Download

 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.