Page 1
Construction of genomic and cDNA libraries
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: Construction of genomic and cDNA libraries
Lesson Developer:Dr. Vibha G. Checker
Department/College:Kirori Mal College, University of Delhi
Lesson Reviewer: Dr Manisha Agarwal
Department/College: Genetics, UDSC
Language Editor: Dr Namrata Dhaka
Department/College:Genetics, UDSC
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Page 2
Construction of genomic and cDNA libraries
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: Construction of genomic and cDNA libraries
Lesson Developer:Dr. Vibha G. Checker
Department/College:Kirori Mal College, University of Delhi
Lesson Reviewer: Dr Manisha Agarwal
Department/College: Genetics, UDSC
Language Editor: Dr Namrata Dhaka
Department/College:Genetics, UDSC
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Construction of genomic and cDNA libraries
Institute of Lifelong Learning, University of Delhi
Table of Contents
Chapter: Construction of genomic and cDNA libraries
• Introduction
• Genome and Genomics
• DNA libraries are collection of cloned DNA fragments
• Construction of Genomic library
• How good is the constructed genomic library
• Construction of a cDNA library
• Genetic Selection of recombinants from the library
• Genetic selection by antibiotic resistance
• Genetic selection by blue white screening
• Genetic selection by plaque morphology
• Screening DNA libraries to obtain gene of interest
• Colony Hybridization
• Polymerase Chain Reaction
• Immunological Screening
• Complementation
• An Enzyme Linked Immunosorbent Assay (ELISA)
• Chemical synthesis of DNA Probes and DNA oligonucleotides
• Summary
• Exercise
• Glossary
• References
Page 3
Construction of genomic and cDNA libraries
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: Construction of genomic and cDNA libraries
Lesson Developer:Dr. Vibha G. Checker
Department/College:Kirori Mal College, University of Delhi
Lesson Reviewer: Dr Manisha Agarwal
Department/College: Genetics, UDSC
Language Editor: Dr Namrata Dhaka
Department/College:Genetics, UDSC
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Construction of genomic and cDNA libraries
Institute of Lifelong Learning, University of Delhi
Table of Contents
Chapter: Construction of genomic and cDNA libraries
• Introduction
• Genome and Genomics
• DNA libraries are collection of cloned DNA fragments
• Construction of Genomic library
• How good is the constructed genomic library
• Construction of a cDNA library
• Genetic Selection of recombinants from the library
• Genetic selection by antibiotic resistance
• Genetic selection by blue white screening
• Genetic selection by plaque morphology
• Screening DNA libraries to obtain gene of interest
• Colony Hybridization
• Polymerase Chain Reaction
• Immunological Screening
• Complementation
• An Enzyme Linked Immunosorbent Assay (ELISA)
• Chemical synthesis of DNA Probes and DNA oligonucleotides
• Summary
• Exercise
• Glossary
• References
Construction of genomic and cDNA libraries
Institute of Lifelong Learning, University of Delhi
Introduction
In this chapter, we shall discuss about the ways in which whole genome of an organism can
be fragmented into small segments and cloned into vectors, for various purposes.
Genome and Genomics
Dr. Tom Roderick, a geneticist at the Jackson laboratory, China, coined the term "genomics"
which refers to the study of entire genome of organisms to gain insights about its structure
and function. The term "genome" originated from the German word ‘Genom’, and was first
used by Hans Winkler. Genes present in all thechromosomes of a particular organism
constitute its genome and the study of this total genetic material of an organism or species
is called genomics.The study of genomics involves the use of various techniques of creating
recombinant DNA and DNA sequencing in order to elucidate structure and function of
genomes.
Page 4
Construction of genomic and cDNA libraries
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: Construction of genomic and cDNA libraries
Lesson Developer:Dr. Vibha G. Checker
Department/College:Kirori Mal College, University of Delhi
Lesson Reviewer: Dr Manisha Agarwal
Department/College: Genetics, UDSC
Language Editor: Dr Namrata Dhaka
Department/College:Genetics, UDSC
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Construction of genomic and cDNA libraries
Institute of Lifelong Learning, University of Delhi
Table of Contents
Chapter: Construction of genomic and cDNA libraries
• Introduction
• Genome and Genomics
• DNA libraries are collection of cloned DNA fragments
• Construction of Genomic library
• How good is the constructed genomic library
• Construction of a cDNA library
• Genetic Selection of recombinants from the library
• Genetic selection by antibiotic resistance
• Genetic selection by blue white screening
• Genetic selection by plaque morphology
• Screening DNA libraries to obtain gene of interest
• Colony Hybridization
• Polymerase Chain Reaction
• Immunological Screening
• Complementation
• An Enzyme Linked Immunosorbent Assay (ELISA)
• Chemical synthesis of DNA Probes and DNA oligonucleotides
• Summary
• Exercise
• Glossary
• References
Construction of genomic and cDNA libraries
Institute of Lifelong Learning, University of Delhi
Introduction
In this chapter, we shall discuss about the ways in which whole genome of an organism can
be fragmented into small segments and cloned into vectors, for various purposes.
Genome and Genomics
Dr. Tom Roderick, a geneticist at the Jackson laboratory, China, coined the term "genomics"
which refers to the study of entire genome of organisms to gain insights about its structure
and function. The term "genome" originated from the German word ‘Genom’, and was first
used by Hans Winkler. Genes present in all thechromosomes of a particular organism
constitute its genome and the study of this total genetic material of an organism or species
is called genomics.The study of genomics involves the use of various techniques of creating
recombinant DNA and DNA sequencing in order to elucidate structure and function of
genomes.
Construction of genomic and cDNA libraries
Institute of Lifelong Learning, University of Delhi
DNA libraries are collection of cloned DNA fragments
Source:https://www.youtube.com/watch?v=SvjeCxVu2dI
DNA cloning is a technique to construct recombinant DNA molecules. Desired DNA
fragmentis inserted with a vector molecule which serves as a means to propagate the
cloned DNA within the cell. DNA library is a large collection of all the sequences from a
specific cell/ tissue/ organism cloned into a suitable vector. Just like a book library is a
collection of books where specific books for varioussubjects can be searched, researchers
Page 5
Construction of genomic and cDNA libraries
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: Construction of genomic and cDNA libraries
Lesson Developer:Dr. Vibha G. Checker
Department/College:Kirori Mal College, University of Delhi
Lesson Reviewer: Dr Manisha Agarwal
Department/College: Genetics, UDSC
Language Editor: Dr Namrata Dhaka
Department/College:Genetics, UDSC
Lesson Editor: Dr Rama Sisodia, Fellow in Botany ILLL
Construction of genomic and cDNA libraries
Institute of Lifelong Learning, University of Delhi
Table of Contents
Chapter: Construction of genomic and cDNA libraries
• Introduction
• Genome and Genomics
• DNA libraries are collection of cloned DNA fragments
• Construction of Genomic library
• How good is the constructed genomic library
• Construction of a cDNA library
• Genetic Selection of recombinants from the library
• Genetic selection by antibiotic resistance
• Genetic selection by blue white screening
• Genetic selection by plaque morphology
• Screening DNA libraries to obtain gene of interest
• Colony Hybridization
• Polymerase Chain Reaction
• Immunological Screening
• Complementation
• An Enzyme Linked Immunosorbent Assay (ELISA)
• Chemical synthesis of DNA Probes and DNA oligonucleotides
• Summary
• Exercise
• Glossary
• References
Construction of genomic and cDNA libraries
Institute of Lifelong Learning, University of Delhi
Introduction
In this chapter, we shall discuss about the ways in which whole genome of an organism can
be fragmented into small segments and cloned into vectors, for various purposes.
Genome and Genomics
Dr. Tom Roderick, a geneticist at the Jackson laboratory, China, coined the term "genomics"
which refers to the study of entire genome of organisms to gain insights about its structure
and function. The term "genome" originated from the German word ‘Genom’, and was first
used by Hans Winkler. Genes present in all thechromosomes of a particular organism
constitute its genome and the study of this total genetic material of an organism or species
is called genomics.The study of genomics involves the use of various techniques of creating
recombinant DNA and DNA sequencing in order to elucidate structure and function of
genomes.
Construction of genomic and cDNA libraries
Institute of Lifelong Learning, University of Delhi
DNA libraries are collection of cloned DNA fragments
Source:https://www.youtube.com/watch?v=SvjeCxVu2dI
DNA cloning is a technique to construct recombinant DNA molecules. Desired DNA
fragmentis inserted with a vector molecule which serves as a means to propagate the
cloned DNA within the cell. DNA library is a large collection of all the sequences from a
specific cell/ tissue/ organism cloned into a suitable vector. Just like a book library is a
collection of books where specific books for varioussubjects can be searched, researchers
Construction of genomic and cDNA libraries
Institute of Lifelong Learning, University of Delhi
use a DNA library to isolate specific genes. DNA libraries are important source of DNA for
genome sequencing, gene identificationand functional analysis.Broadly two different types
of DNA libraries can be constructed depending upon the source of starting DNA:
(A) Genomic library
(B) cDNA library
When the source of starting DNA is complete genomic DNA of a particular organism, the
DNA library is called a genomic library. It therefore representsat least one copy of every
gene in an organism’s genome. Theoretically,the genomic DNA is digested with restriction
enzymes and all the generated fragments are cloned into suitable vectors and these vectors
are transferred to host cells by transformation. These transformed cells represent genomic
library,i.e. collection of atleast one copy of all the sequences present in the genome.
This type of library can be used for many purposes:
? To generate DNA for sequencing a genome,
? To investigate the structure of a given chromosome,
? To study the untranslated regions of a gene including promoter or introns and
? To clone smaller genes.
The DNA sequences in this type of library contain coding and non-coding sequences;
therefore these genomic libraries are more suited to represent the genetic constitution of
simpler organisms like bacteria or yeast. The genes of higher eukaryotes contain intron
sequences (usually larger than exons), which makes genes of higher eukaryotes too large to
be inserted intact into vectors. Consequently, the complete DNA sequence of a given gene is
represented in more than one clone. Many a times, DNA inserts in cloning vectors do not
contain sequence of coding region of the DNA but contain non coding region of the gene,as
a result it becomes difficult to identify coding regions of a gene that encodes protein
sequences.
Read More