Genetic Engineering
Genetic Engineering
• Commercially available
• EcoRI - Escherichia (E) coli (co), strain Ry13 (R)
and first endonuclease (I).
• Hindlll
Types
• Annealing
• Addition of oligo (dA) to 3′-ends of some DNA
molecules and the addition of oligo (dT) to 3′-
ends of other molecules.
• The homo-polymer extensions (by adding 10-40
residues) can be synthesized by using terminal
deoxynucleotidyltransferase (of calf thymus).
• Homo-polymer tailing, achieved by annealing
Linkers, Adaptors
• Escherichia coli:
• organism used in the DNA technology.
• Undoubtedly, E.coli, the simplest Gram
negative bacterium (a common bacterium of
human and animal intestine), has played a key
role in the development of present day
biotechnology
• E.coli can double in number every 20 minutes.
• Plasmids (along with foreign DNA) also
multiply to produce millions of copies, -
• Colony or in short clone.
• Clone is broadly used to a mass of cells,
organisms or genes that are produced by
multiplication of a single cell, organism or
gene.
Bacillus subtilis:
• Viruses
• DNA gets incorporated into the bacterial
chromosome and remains there permanently.
• Phage vectors can accept short fragments of
foreign DNA into their genomes.
• Hence phage vectors are preferred for working
with genomes of human cells.
• Phage λ consists of a head and a tail (both
being proteins) and its shape is comparable to
a miniature hypodermic syringe.
• The DNA, located in the head, is a linear
molecule of about 50 kb.
• At each end of the DNA, there are single-
stranded extensions of 12 base length each,
which have cohesive (cos) ends.
• The phage DNA has two fates
• Lytic cycle
• Lysogenic cycle.
Bioreactors
• Electroporation:
• Electroporation is based on the principle that high
voltage electric pulses can induce cell plasma
membranes to fuse.
• Electric field-mediated membrane permeabilization.
• Electric shocks can also induce cellular uptake of
exogenous DNA (believed to be via the pores formed
by electric pulses) from the suspending solution.
• Simple and rapid technique for introducing
genes into the cells from various organisms
(microorganisms, plants and animals).
• The cells are placed in a solution containing
DNA and subjected to electrical shocks to
cause holes in the membranes.
• The foreign DNA fragments enter through the
holes into the cytoplasm and then to nucleus.
Liposome-Mediated Gene Transfer