Overview of Basic Tools and Methods for Genetic Engineering E-Book

Manoj Parakhia

• Herausgeber: GRIN Verlag
• Sprache: Englisch

Veröffentlichungsjahr: 2015

Beschreibung

Abstract from the year 2015 in the subject Biology - Genetics / Gene Technology, Junagadh Agricultural University (Department of Biotechnology), language: English, abstract: 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.

Leseprobe

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Index

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.

1. GENE CLONING PROCEDURES

 The recombinant DNA technology involves the cloning of the higher organisms DNA into the prokaryotic organisms so as to allow these lower organisms to produce the specific product coded in the gene of higher organisms.

 The recombinant DNA technology is simply a “cut-paste mechanism” where a single gene is isolated from one organism (cut) and Inserted to another organism (paste) that will produce the new product or character (phenotype) according to the message (genotype) present in the gene.

 Among many enzymes, the most important are the enzymes used for cleaving the DNA, these enzymes are called Restriction Endonucleases (REN).

 These enzymes are the enzymes that have the capacity to break the DNA from phospho-diester bond between the two adjacent nucleotides.

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.