unit 1 genetic engineering
TRANSCRIPT
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UNIT-1Gene Cloning
Presented By:-Ms. Smita Shukla
Assistant ProfessorN.I.E.T, Greater Noida
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What is a Gene ?
• Gene is a hereditary unit consisting of a sequence of DNA that occupies a specific location on a chromosome and determines a particular characteristic in an organism.
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WHAT IS CLONING ?• Cloning in Biotechnology refers to processes
used to create identical copies of DNA fragments , cells or organisms .
* Clone is a group of cells derived from a single
parental cell by asexual reproduction
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These identical copies are called clones
Cloning refers to DNA fragments - molecular or gene cloning
Cloning refers to cells – cell cloning
Cloning refers to organism - organism cloning
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What is gene cloning ?• To "clone a gene" is to make
many copies of it
• Act of making many identical copies of gene
• Gene can be an exact copy of a natural gene
• Gene can be an altered version of a natural gene
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Whole organisms are cloned too, but differently
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BASIC STEPS IN GENE CLONING
• Preparation of Pure sample of DNA from the organism of desire
• Cutting of the DNA molecule using Restriction enzymes to form fragments
• Analysis of DNA fragments using electrophoresis
The action of a restriction enzyme ,EcoRI
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Steps in Cloning a Gene• The First step in cloning a gene is to isolate the
DNA from the organism that contains the desired gene.
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Steps in Cloning a Gene• The isolated DNA is purified and then
fragmented with a restriction enzyme.
• Restriction enzymes used in cloning produce staggered cuts in specific sequences in the DNA, generating fragments with the cohesive ends.
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Steps in Cloning a Gene
• Each fragment has a single stranded sequence of nucleotides on its ends that is capable of hybridizing with DNA that has been fragmented with the same restriction enzyme.
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• The DNA fragments are then incorporated into the plasmids.
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• The type of plasmid used for cloning has a single restriction site, and when cleaved by the restriction enzyme, generates the same cohesive ends that are in the fragments of DNA to be cloned.
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• The cohesive ends of the plasmid and DNA fragments now line up and the enzyme, DNA ligase, is used to form Phosphodiester bonds.
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• The next step is to incorporate the plasmid into bacterial host cells by transformation.
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• Each cell contains a different segment of DNA from the original organism. Taken together these cells represent a DNA library.
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• The cells can now be plated out on agar medium.
• The colony of cells containing the desired cloned gene can then be identified and isolated.
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INFERENCE• Within the host cell the vector multiplies and
produces numerous identical copies not only of itself but also of the gene that it carries.
• After that the host cell divides … as the recombinant vector replicates
• Now colony or clone, of identical host cells is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule
• The gene is cloned.
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Application of Bacteria & Virus used in Genetic
Engineering
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What is genetic engineering?
• Genetic engineering refers to the direct manipulation of DNA to alter an organism’s characteristics in a particular way.
• This may mean changing one base pair (A-T or C-G), deleting a whole region of DNA, or introducing an additional copy of a gene.
• It may also mean extracting DNA from another organism’s genome and combining it with the DNA of that individual.
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• A gene is much too small to be inserted with some kind of microsurgery.
• Therefore some carrier ("vector") is required which takes the gene into the recipient cell and somehow gets it inserted into the DNA of the recipient.
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Methods for gene insertion• Different means are used for carrying the desired
gene into the hereditary substance..
• For plants, the most common method is the use of a bacterium, Agrobacterium tumefaciens.
• For animals, certain viruses are used.
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1. Bacteria as gene carriers
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PLASMIDS
A small circles of DNA found in bacteria andsome other organisms.• Plasmids can replicate independently of the
host cell .
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Desirable properties of a Vector
• It should be small.• Its DNA sequence should be known.• It should grow to high copy number in the host
cell.• It should contain a selectable marker that allows
easy selection of transformed host cell.• It should replicate autonomously• Should be easy to isolate & purify• Should contain one unique restriction enzymes.
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• Also, when expression of DNA is needed then expression vectors are used.
• Expression vectors should contain control elements; promoter, operator etc..
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Types of vectors• Choice of vector is dependent on insert size and
application.
• For Bacterial Cells:• Plasmids• Bacteriophage( Lambda phage & M 13 Phage)• Cosmids• Phagemids• YAC( Yeast Cloning Vectors)
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For Plant cells:Binary Vector systemCo -integrative vector system
For Animal/ Mammalian cells:RetrovirusSV40 Viral VectorEBV( Epstein- Barr Virus), BPV( Bovine Papilloma
Virus)
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Cloning VectorContains: • An origin of replication
• Selectable marker gene ( eg. Ampicillin resistance gene) and
• Multiple cloning site containing unique restriction enzymes sites..
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Plasmid• A small circular DNA molecule found in
bacteria.• Used as cloning vectors. • Example: PUC19
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Plasmid classification• Fertility or F – plasmids
• Resistance or R plasmids
• Col plasmids
• Degradative plasmids
• Virulence
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2. Bacteriophages
• Phages are very simple in structure,
• Consisting of a DNA molecule carrying a number of genes, including several for replication of the phage, surrounded by a protective coat or capsid made up of protein molecules
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• Examples: T2, T4 and T6 Bacteriophages that infect E.coli.
• Filamentous phages with single stranded DNA such as M13
• Transfer of genetic elements from one bacterium to another by a bacteriophage is termed as transduction.
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Why is Bacteria used for genetic engineering?
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• Generation time is very small that is 20 minutes.
• Bacteria's DNA is very simple and can easily be transplanted from one bacterium to another
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Cloning Process
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How are viruses used in Genetic Engineering
• Viruses are tiny, nonliving particles that must have a host cell to replicate and flourish.
• Ability to attach to and invade specific cells, and incorporate the DNA (and/or RNA) they are carrying into the host cell, where it combines with the host cell's DNA.