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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
FAQs on Lecture 14 - Construction of Genomic and cDNA Libraries - Plant Biotechnology - Botany
| 1. What is a genomic library? |  |
Ans. A genomic library is a collection of DNA fragments that represent the entire genome of an organism. It is created by cutting the genomic DNA into fragments and inserting them into a vector, such as a bacterial artificial chromosome (BAC) or a phage. This library allows researchers to study and analyze specific genes or sequences of interest.
| 2. What is a cDNA library? | |
Ans. A cDNA library is a collection of complementary DNA (cDNA) fragments that are synthesized from mRNA molecules. It represents the expressed genes in a specific cell or tissue at a given time. The cDNA library is created by reverse transcribing mRNA into cDNA and then inserting it into a vector. This library is useful for studying gene expression patterns and identifying genes that are active in specific conditions.
| 3. How are genomic libraries constructed? | |
Ans. Genomic libraries are constructed by isolating genomic DNA from the organism of interest and then cutting it into smaller fragments using restriction enzymes. These fragments are then inserted into a vector, such as a BAC or a phage, which can be propagated in bacteria. The vectors are then introduced into host cells, such as bacteria, to create a library of clones containing different genomic fragments. This library can be used for various applications, such as gene mapping or sequencing.
| 4. What are the advantages of using cDNA libraries? | |
Ans. Using cDNA libraries has several advantages. Firstly, cDNA libraries only contain sequences that are actively transcribed, allowing researchers to focus on genes that are expressed in specific conditions or tissues. Secondly, cDNA libraries can provide information about gene expression patterns and help identify genes involved in specific biological processes. Lastly, cDNA libraries can be used to study alternative splicing and post-transcriptional modifications of genes.
| 5. How are genomic and cDNA libraries used in botany research? | |
Ans. Genomic and cDNA libraries are extensively used in botany research. Genomic libraries help in identifying and characterizing genes involved in various plant processes, such as growth, development, and response to environmental stimuli. They can also be used for genetic mapping and marker development. On the other hand, cDNA libraries are used to study gene expression patterns, identify genes involved in specific plant pathways or responses, and study the regulation of gene expression in different plant tissues or under different conditions. Both types of libraries are valuable tools for understanding the genetic basis of plant biology.
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