genetic engineering
TRANSCRIPT
What is genetic engineering
• Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype.
• Term “Genetic Engineering” was coined by Jack Williamson.
• In 1972, Paul berg created the first recombinant DNA molecules by combining DNA from the monkey virus SV40 with that of the lambda virus.
• In 1973 Herbert Boyer and Stanley Cohen created the first transgenic organism by inserting antibiotic resistance genes into the plasmid of an E. coli bacterium.
• The first trials of genetically engineered plants occurred in France and the USA in 1986, tobacco plants were engineered to be resistant to herbicides.
• The Flavr Savr tomato was a tomato engineered to have a longer shelf life. In 1995, Bt Potato was approved safe by the Environmental Protection Agency.
• Bt-Cotton is a genetically modified cotton which is resistant to pests. Golden Rice genetically modified to contain beta-carotene(a source of Vitamin A).
Enzymes: Restriction Endonucleases
• Enzymes that cut DNA• Each has a known
sequence of 4 to 10 pairs as its target
• Can recognize and clip at palindromes– Madam, I’m Adam– GAATTC– CTTAAG
Restriction endonucleasemakes staggered cutat palindrome.
(1)
(2)
Sticky ends
Action of restriction endonucleases. (1) A restriction endonucleaserecognizes and cleaves DNA at the site of a specific palindromic sequence. Cleavage can produce staggered tails called sticky ends that accept complementary tails for gene splicing. (2) The sticky ends can be used to join DNA from different organisms by cutting it with the same restriction enzyme, ensuring that all fragments have complementry ends.
(c)
DNAOrganism
2
DNAOrganism
1
Site of cut
G A T C
C T A G
G A T CC T A G
TGC
A
ACGT
TGCA
ACGT
TGC
A
ACGTTGCA
ACGT
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Some Common Restriction Enzymes
• BamHI - Bacillus amyloliquefaciens G|GATCC
CCTAG|G
• EcoRI - Escherichia coliG|AATTC
CTTAA|G
• EcoRV - Escherichia coliGAT|ATC
CTA|TAG
Enzymes: Ligase and Reverse Transcriptase
– Ligase: Enzyme necessary to seal sticky ends together.
– Reverse transcriptase: enzyme that is used when converting RNA into DNA.• Copied DNA is referred to as complementary DNA or cDNA
Vectors
Vectors are autonomous replicating DNA molecule it may be natural one (plasmids & Bacteriophage) or artificial once (cosmids, YAC & BAC) used to carry desired gene from donor cell to host cell for its cloning is called vector.
Properties of a good vector
• It should be able to replicate autonomously.• A vector should be ideally less than 10 kb in size.• Vector should be easy to isolate & purify.• It should be easily introduced into the host cells.• The vector should have suitable marker genes.• Vector should contain multiple cloning site.• It should have the ability to integrate either itself or the
DNA insert it carries into the genome of the host cell.• It should contain at least suitable control elements viz
promoter, operator & ribosome binding sites.
Properties of a Good Host
• Should be easy to transform• Should support the replication of rDNA • Should be free from elements that interfere replication of
rDNA • Lack the active restriction enzyme Eg. E. coli strain HB 101.• Should not have methylase enzyme that methylate the
DNA, that become resistant to use full restriction enzymes.• Should deficient in normal recombination function, so that
DNA insert is not altered by recombination.
Types of vectors
Cloning vector 1. Plasmid vector2. Bacteriophage vector3. Cosmid vector4. Phagemid vector 5. Phasmid vector6. Artificial chromosomes
Expression vector
Cloning vectorA vector used for transfer and multiplication of desired DNA in
suitable host is called as cloning vector.
Plasmid: The extra chromosomal, self replicating, double stranded & circular DNA molecules of bacteria that carries the characters like sex factor, drug resistance, colicin factor etc. is called as Plasmid.
Cosmid are essentially plasmids that contain minimum of 250 bp of lambda DNA.
Phagemid: A plasmid vector that contains origin of replication from a phage, in addition to that of plasmid.
Phasmid: a gene-cloning vector consisting of an artificial combination of a plasmid with a phage such that its genome contains functional origins of replication of both; it may thus be propagated either as a plasmid or as a phage in appropriate host strains.
Process of Genetic Engineering
Five steps involved in this process:1. Isolation
2. Cutting
3. Insertion (Ligation)
4. Transformation
5. Expression
Step 1: Isolating the gene
• Proteins called “restriction enzymes” are used to cut the DNA into small pieces.
Step 2: Inserting gene into vector
• Vector – molecule of DNA which is used to carry a foreign gene into a host cell.
• Researches have succeeded in disrupting the tumor causing genes and inserting new genes for resistance to disease, frost and herbicides.
Basic steps in transformation of plant cells by Agrobacterium tumefaciens.
• The Ti plasmid can be removed from the agrobacterium, cut open with a restriction endonuclease and mixed with DNA from another source (foreign DNA) that has been digested with the same restriction enzyme.
The plasmid can then be put back into agrobacterium
by transformation. Agrobacterium attaches to the plant cell and a copy of the
plasmid is transferred into the plant cell.
• When a plant cell divides, each daughter cell receives
the new gene.• In some cases the entire plant with the new trait can be
generated from a single cell.
Disadvantages of GE
• Modified genes can have an irreversible effects and associated consequences.
• Unknown Consequences of Viral Genes• Uncertain Effects that may be brought by genetically
modified life form.