The Basic Principles of Gene Cloning and DNA Analysis
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Gene cloning is one of the most important techniques in biotechnology and modern molecular biology. It is the same technique that was used in the Human Genome Project that successfully determined the sequences of almost all the genetic content of the chromosomes of humans. Moreover, the very first commercially available recombinant product “Insulin” was also the result of gene cloning. The technique has been used in a wide variety of studies and has great importance from the industrial point of view as well. It is, thus, important to have an idea of what gene cloning is and how it works.
This course describes the basic principles of gene cloning in a simple way and is based on the book “Gene Cloning and DNA analysis” by T.A. Brown. The course has a number of lectures on topics such as vectors used for gene cloning, purification of DNA, synthesis of recombinant DNA molecules, the introduction of DNA into living cells, selection of the transformed cells, selection of the desired recombinants, polymerase chain reaction and DNA analysis using gel electrophoresis. Each section/module has at least 1 Quiz in order to test the knowledge that you’ll gain in the respective section/module. Apart from lectures and quizzes, the course consists of a mid-term and a final term; each of which consists of a practice test and an assignment. Practice tests are of particular importance as, unlike quizzes, they are not based on the knowledge but on the application of the knowledge that you’ll gain in this course.
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Gene CloningAt the end of this section, students will have a very basic understanding of gene cloning.
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Polymerase Chain Reaction
At the end of this section, students will have a very basic understanding of PCR.
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Obtaining a Pure Sample of a Gene by Cloning
At the end of this lecture, students will have an understanding of the use of gene cloning to obtain a pure sample of DNA.
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Obtaining a Pure Sample of a Gene by PCR
At the end of this lecture, students will have an understanding of the use of PCR to obtain a pure sample of DNA and the certain limitations of PCR.
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Gene Cloning and PCRThis quiz consists of questions from the lectures of Section 2 and will help you to get an idea of how much you have learned in these lectures.
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Vectors for Gene Cloning: Introduction
At the end of this lecture, students will have a very basic understanding of vectors and their few characteristics.
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Plasmids: Basic Characteristics and Replication
At the end of this lecture, students will have enough understanding of a plasmid and its characteristics and replication. Moreover, they'll be able to differentiate between:
Non-integrative plasmids and episomes
Stringent and relaxed plasmids
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Plasmids: Classification and Examples
At the end of this lesson, students will have a piece of basic knowledge of five common classes of naturally occurring plasmids of bacteria.
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Cloning Vectors Based on E. coli Plasmids
At the end of this lesson, students will have enough information about pBR322 and pUC8 vectors. Moreover, they'll also get to know about the basic components that a cloning vector should have.
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Plasmids and Plasmid-Based Vectors
This quiz consists of questions from the first four lectures of Section 3 and will help you to get an idea of how much you have learned in these lectures.
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Bacteriophages and Their Life Cycle
At the end of this lecture, students will be able to differentiate between:
Bacteriophage and other viruses
Lytic cycle and lysogenic cycle
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Lambda Phage and M13 Phage
At the end of this lecture, students will have basic information about the lambda phage and M13 phage, their mode of replication, and the nature of their genomes.
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Lambda Bacteriophage Based Vectors
At the end of this lecture, students will be able to understand the problems that arise regarding the packaging of the recombinant vector (based on the phage genome) and will be able to use natural selection as a method of reduction or removal of multiple sites for a particular restriction enzyme in the phage genome. They'll also have enough information regarding insertion vectors, replacement vectors, and cosmids; and regarding cloning using these vectors.
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M13 Based Vectors
At the end of this lesson, students will have enough information about M13-based vectors, will be able to understand the importance of these vectors, and will be able to differentiate M13-based vectors from lambda bachteriphage-based vectors.
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Vectors for Cloning in Yeast
At the end of this lesson, students will have enough information about the vectors used for gene cloning in the yeast Saccharomyces cerevisiae and will be able to differentiate among yeast episomal plasmids (YEps), yeast integrative plasmids (YIps), and yeast replicative plasmids (YRps). Moreover, they'll be able to design a cloning experiment involving yeast using these vectors.
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Bacteriophages, Bacteriophage-Based Vectors, and Vectors for Cloning in Yeast
This quiz consists of questions from the last five lectures of Section 3 and will help you to get an idea of how much you have learned in these lectures.
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DNA Purification: Introduction
At the end of this lesson, students will be aware of different kinds of DNA molecules that may be required in a gene cloning or genetic engineering experiment.
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Genomic DNA Purification from Bacteria
At the end of this lesson, students will be able to extract, isolate, and purify the genomic DNA or total cellular DNA from the bacterial cells.
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Genomic DNA Preparation from Non-Bacterial Organisms
At the end of this lesson, students will be able to extract, isolate, and purify the genomic DNA or total cellular DNA from animal and plant cells.
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Preparation of Genomic DNA from Bacteria and Non-Bacterial Organisms
This quiz consists of questions from the first three lectures of Section 4 and will help you to get an idea of how much you have learned in these lectures.
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Preparation of Plasmid DNA
At the end of this lesson, students will be able to extract, isolate, and purify the plasmids from the bacterial cells.
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Preparation of Lambda Phage DNA
At the end of this lesson, students will be able to extract, isolate, and purify the DNA from the lambda phage and will also become aware of the problems that may be encountered particularly during the step where a large number of lambda phage particles are intended to be obtained.
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Preparation of M13 Phage DNA
At the end of this lesson, students will be able to extract, isolate, and purify the DNA from the M13 phage.
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Preparation of Plasmid and Phage DNA
This quiz consists of questions from the last three lectures of Section 4 and will help you to get an idea of how much you have learned in these lectures.
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Mid Term
This practice test will help students to apply all the knowledge they have gained in the previous lectures in order to solve real-life problems associated with gene cloning, polymerase chain reaction, vectors for gene cloning, and DNA isolation and purification. The purpose of this practice test is to evaluate the amount of knowledge gained by the students as well as their critical thinking ability.
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Cloning Vectors
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DNA Manipulation: Introduction
At the end of this lecture, students will become aware of the basic DNA manipulations and the basic DNA manipulative enzymes.
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Nucleases
At the end of this lecture, students will be able to differentiate between
Endonucleases and exonucleases
Different types of exonucleases
Different types of endonucleases
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Restriction Endonucleases
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Ligases and Ligation
At the end of this lecture, students will have enough information about ligases, their mode of action, and their role in a gene cloning experiment. Moreover, they'll also be able to determine the difference between sticky end and blunt end ligation and will become aware of the problems associated with blunt end ligation.
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Nucleases and Ligases
This quiz consists of questions from the first four lectures of Section 5 and will help you to get an idea of how much you have learned in these lectures.
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Adding Sticky Ends to the Blunt Ends
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DNA Polymerases
At the end of this lecture, students will become aware of a number of DNA polymerases that are used in gene cloning experiments including DNA polymerase I, Klenow polymerase, and Reverse transcriptase.
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DNA Modifying Enzymes
At the end of this lecture, students will have enough knowledge about different DNA modifying enzymes used in the gene cloning experiment and their mode of action.
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Adding Sticky Ends to Blunt Ends, Polymerases, and DNA modifying enzymes
This quiz consists of questions from the last three lectures of Section 5 and will help you to get an idea of how much you have learned in these lectures.
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Introduction of Recombinant DNA Molecule into Living Cells
In this lecture, students will get an outline of the topics that are discussed in this module/section.
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Introduction of Plasmid-Based Vectors in Bacteria - Part I
At the end of this lecture, students will have enough knowledge about
The introduction of DNA into bacteria
Different products that are produced during ligation.
The selection of the transformants when pBR322 is used as a cloning vector.
The selection of the cells having the recombinant vector when pBR322 is used as the cloning vector.
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Introduction of Plasmid-Based Vectors in Bacteria - Part II
At the end of this lecture, students will have enough knowledge about
The selection of the transformants when pUC8 is used as a cloning vector.
The selection of the cells having the recombinant vector when pUC8 is used as the cloning vector.
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Introduction of Recombinant Phage DNA into Bacteria
At the end of this lecture, students will have enough knowledge about
Different methods of introduction of recombinant phage DNA into bacteria.
Methods used to obtain the packaging proteins of the lambda phage for in vitro packaging of the phage DNA.
Strategies used for the identification of the recombinant phages.
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Introduction of DNA into Non-Bacterial Cells
At the end of this lecture, students will have enough knowledge about
The introduction of DNA into animal cells.
The introduction of DNA into plant cells.
The introduction of DNA into yeast and fungal cells.
Physical methods of DNA introduction
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Introduction of DNA into Living Cells
This quiz consists of questions from the lectures of Section 6 and will help you to get an idea of how much you have learned in these lectures.
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Obtaining Clone of A specific Gene: Introduction
In this lecture, students will get introduced to:
The problem of selection of the desired recombinant clone.
The two strategies that can be used to identify the desired recombinant clones.
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Direct Selection
At the end of this lecture, students will be able to design a cloning experiment in which the selection of the desired recombinant clones can be carried out using the direct selection strategies.
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Construction of Libraries
In this lecture, students will get introduced to the genomic and cDNA libraries and to the procedure of obtaining the genomic and cDNA libraries.
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Methods of Clone Identification Based on DNA Probes
In this lecture, students will get an idea of how we can use the labeled DNA probes to identify the desired recombinant clones from the genomic or cDNA libraries.
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Methods of Clone Identification Based on Translation Product
In this lecture, students will get an idea of how the immunological screening can be used to identify the desired recombinant clones from the genomic or cDNA libraries and how we can obtain the antibodies that bind specifically to our target protein encoded by our gene of interest.
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Obtaining Clone of a Specific Gene
This quiz consists of questions from the lectures of Section 7 and will help you to get an idea of how much you have learned in these lectures.
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Gel Electrophoresis
In this lecture, students will get to know:
The principle of electrophoresis
The principle of gel electrophoresis
The difference between electrophoresis and gel electrophoresis
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Visualizing Bands of DNA Molecules in an Agarose Gel
In this lecture, students will learn how we can stain the DNA and visualize the DNA bands as well as how we can use gel electrophoresis to predict the size of the DNA molecules in our test sample.
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Gel Electrophoresis
This quiz consists of questions from the lectures of Section 8 and will help you to get an idea of how much you have learned in these lectures.
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Polymerase Chain Reaction: Introduction
In this lecture, students will get introduced to
Polymerase chain reaction
Different steps of PCR
The typical temperature profile of a PCR
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Primers for PCR
In this lecture, students will get introduced to
Forward and reverse primers
Characteristics of the primers
Factors critical for primer specificity
Measuring the annealing temperature of a primer
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Analyzing and Cloning PCR Products
At the end of this lecture, students will have enough knowledge about the analysis of the PCR product using gel electrophoresis and the cloning of the PCR products.
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Quantitative PCR
In this lecture, students will get introduced to quantitative PCR (qPCR) and will get an idea of how we can use the qPCR to quantify the DNA.
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Polymerase Chain Reaction
This quiz consists of questions from the lectures of Section 9 and will help you to get an idea of how much you have learned in these lectures.
