This summary provides key words as an overview of basic tools and methods used to modify genetic material. The manipulation of genetic material requires the knowledge of basic tools and procedures used for the manipulation of the genome. The aim of the text is to help understand some processes of genetic engineering.
Table of Contents
1. GENE CLONING PROCEDURES
1.1 RESTRICTION ENDONUCLEASES
1.2 ISOLATION OF DNA TO BE CLONED
1.3 C-DNA SYNTHESIS
1.4 GENE LIBRARY
1.5 RECENT TECHNIQUE
1.6 VARIOUS VECTORS USED IN r-DNA TECHNOLOGY
1.7 PLASMID CLONING VECTOR
1.8 CONSTRUCTION OF pBR322
1.9 VIRAL DNA / BACTERIOPHAGES AS VECTOR
1.10 COSMIDS AS VECTOR
1.11 JOINING OF THE DNA WITH VECTOR
1.12 TRANSFORMATION AND GROWTH OF CELL
1.13 SELECTION OF CLONES
1.14 EXPRESSION OF CLONED DNA
1.15 PROTEIN PROTECTION
2. GENETIC MANIPULATION OF EUKARYOTIC CELLS
2.1. GENETIC MANIPULATION OF PLANT CELLS
2.2 GENETIC MANIPULATION IN MAMMALIAN CELL.
2.3 GENETIC MANIPULATION IN YEAST
3. BLOTTING TECHNIQUES
3.1. ANALYSIS OF DNA BY SOUTHERN BLOTTING
3.2 ANALYSIS OF PROTEIN BY WESIERN BLOT TECHNIQUES.
4. DNA SEQUENCING
4.1 DNA SEQUENCING BY CHEMICAL DEGRACATION METHOD.
4.2 SEQUENCING BY CHAIN TERMINATION
4.3 DIRECT DNA SEQUENCING BY USING PCR.
Objectives and Research Themes
This book explores the fundamental principles and practical methodologies of genetic engineering, specifically focusing on the recombinant DNA technology required to transfer and express genes from higher organisms into prokaryotic hosts. It aims to clarify the complex processes of DNA isolation, cloning vector construction, and the strategic manipulation of eukaryotic cells to achieve successful gene expression and protein production.
- Techniques for DNA cloning and the application of restriction endonucleases.
- Comparative analysis of different cloning vectors including plasmids, bacteriophages, and cosmids.
- Methodologies for the genetic manipulation of plant, mammalian, and yeast cells.
- Advanced analytical procedures such as blotting techniques and DNA sequencing methods.
Excerpt from the Book
1.1 RESTRICTION ENDONUCLEASES
REN are the enzymes produced normally by various types of bacteria as a weapon against invading viruses. These enzymes can cleave the viral DNA and therefore they can restrict the further growth of viruses in that bacterium. Due to this character, they are called Restriction Endonucleases.
Their existence was first identified by Werner Arber in early 1960s when he was studying on bacteriophages.
Arber along with two other scientists Daniel Nathans and Hamilton Smith was awarded Nobel Prize in 1978 for their work with restriction enzymes.
There are basically three types of REN found in nature…
Type I REN
Type II REN
Type III REN
Among these three types, Type II REN are most commonly used during gene cloning processes. Their cleavage is site specific.
Their molecular weight is in the range of 20000-100000 Da. They contain two identical subunits.
These enzymes can cut both the strands of DNA after recognizing specific nucleotide sequence.
Summary of Chapters
1. GENE CLONING PROCEDURES: Covers the basic mechanism of DNA cloning, including the isolation of DNA, the role of restriction enzymes, and strategies for cloning DNA into vectors.
2. GENETIC MANIPULATION OF EUKARYOTIC CELLS: Discusses the challenges of using bacteria and details alternative strategies for manipulating plant, mammalian, and yeast genomes.
3. BLOTTING TECHNIQUES: Describes methods for identifying and characterizing DNA, RNA, and protein products using Southern, Northern, and Western blotting.
4. DNA SEQUENCING: Outlines various methodologies for determining the nucleotide sequence of DNA, ranging from chemical degradation to PCR-based approaches.
Keywords
Genetic Engineering, Recombinant DNA Technology, Restriction Endonucleases, Gene Cloning, Plasmid Vectors, Bacteriophages, Cosmids, Blotting Techniques, Southern Blot, Western Blot, DNA Sequencing, PCR, Gene Expression, Protein Protection, Eukaryotic Cells
Frequently Asked Questions
What is the fundamental purpose of this work?
The book provides a comprehensive technical guide to the principles of genetic engineering, detailing how scientists clone and express foreign DNA in host organisms.
What are the central thematic areas covered?
Key themes include recombinant DNA methodology, the selection and construction of vectors, genetic modification of different cell types, and analytical identification techniques.
What is the primary goal of the recombinant DNA processes described?
The primary goal is to allow lower organisms (like bacteria) to produce specific products encoded by genes derived from higher organisms.
What primary scientific methods are employed in these cloning processes?
The book details the use of restriction enzymes, DNA ligation, transformation, colony hybridization, and various blotting and sequencing techniques.
What is the focus of the book's main section?
The main section focuses on the practical aspects of gene cloning, from initial DNA isolation to the complexities of expressing proteins and ensuring their protection within the host.
Which keywords best characterize this work?
Key terms include Recombinant DNA, Cloning, Restriction Enzymes, Vectors, Gene Expression, and DNA Sequencing.
Why are Ti Plasmids significant in plant cell manipulation?
Ti Plasmids are utilized because they naturally transfer T-DNA into plant chromosomes, making them highly efficient vehicles for plant genetic engineering.
What challenge does the 'protein protection' chapter address?
It addresses the issue of host bacteria degrading or killing eukaryotic proteins, proposing solutions like the attachment of host DNA fragments or the use of protease inhibition genes.
- Citar trabajo
- Dr Manoj Parakhia (Autor), R.S. Tomar (Autor), V.M. Rathod (Autor), B.A. Golakiya (Autor), 2015, Overview of Basic Tools and Methods for Genetic Engineering, Múnich, GRIN Verlag, https://www.grin.com/document/304373
