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© SSER Ltd.
Gene Technology
Gene Technology or Recombinant DNA Technology is about the manipulation of genes
Recombinant DNA Technology involves the isolation of DNA sequences from one
organism and combining them with the DNA from a different organism
This technique allows organisms to express genes and thus manufacture
proteins that they would not normally produce – their DNA has been engineered
to manufacture a product that is foreign to their natural genetic make-up
The procedure of Recombinant DNA Technology involves introducing
segments of DNA (genes) into a HOST by means of a carrier (VECTOR) system
The foreign DNA (gene) becomes a permanent feature of the host such that,
during replication of the host cell, the gene is also passed on to its daughter cells
along with the rest of its own DNA – the foreign gene is therefore cloned
Growth
Hormone
The manufacture of human insulin
and growth hormone are
examples of the
application of Recombinant
DNA Technology
Human
Insulin
The Procedure of Recombinant DNA Technology
Obtain the gene that codes for the desired
protein product
Select a suitable vector (carrier) for transferring the gene to the host cells;
the most commonly used vectors are bacterial plasmids and viruses
Insert the selected gene into the chosen vector
Allow the vector to carry the gene into the host cells; commonly used host cells
are bacteria and yeast cells – they are chosen for their rapid rates
of reproduction and allow the gene to be CLONED
Locate the host cells that have successfully taken up the desired gene
Culture the transformed cells on a commercial scale using large fermenters
Isolate and purify the desired ‘gene product’ – Downstream
Processing
The ‘Toolkit’
The genetic engineer uses five basic ‘tools’ during the
procedure of Recombinant DNA Technology
• Enzymes capable of ‘cutting’ DNA at specific sites – these
enzymes are known as Restriction Enzymes
• Isolated genes that code for the desired product
• Vectors (carriers) into which the desired gene may be
inserted and which are capable of carrying the
gene into a suitable host – bacterial plasmids are commonly
used as vectors
• An enzyme capable of ‘glueing’ an isolated gene into a ‘cut’
vector – DNA ligase is responsible for re-forming the DNA
backbone following insertion of the gene
• A host organism that will take up the vector
containing the gene and reproduce rapidly
in order to supply many copies of the
gene (gene cloning) – the bacterium E. coli
is commonly used as a host
circular
plasmid
plasmid
with
inserted
gene
The ‘Toolkit’
Restriction Enzymes are the engineer’s DNA – cutting scissors
Restriction enzymes, also known as Restriction Endonucleases, are
a group of enzymes found in bacteria that recognise specific DNA
sequences of four to six nucleotides and make their incision within
that sequence
The specific nucleotide sequences, recognised by restriction
enzymes, are called restriction sites and these are usually in the
form of palindromes
Palindromes are nucleotide sequences that are symmetrical, about
an axis, and read the same in opposite directions in the two
strands of DNA
central
enzyme cuts
axis
A restriction enzyme known as
portion of
G A A T T C
Eco R1, makes double-stranded
doublestranded
cuts between the
C T T A A G
DNA
A and G nucleotides on
either side of the central axis
enzyme cuts
The cuts from this enzyme
are staggered and produce
single-stranded regions
called ‘sticky ends’
G
C T
A A
T
A A
T
T
C
G
‘sticky ends’
The ‘Toolkit’
Some restriction enzymes, such as Hpal, cut the DNA strands at positions
directly opposite one another, giving blunt ends to the fragments
Hpal recognises the nucleotide sequence GTTAAC and ‘cuts’ between the
T and A nucleotides about the central axis
enzyme cuts
G T
T
C A A
A A C
T
T
G
enzyme cuts
G T
T
C A A
A A C
T
Over seven hundred different blunt ends
restriction enzymes have now been
identified and isolated from bacterial
cells; each enzyme is named after the
bacterial strain from which it was
derived
T
G
Eco R1 is from Escherichia coli,
strain RY13
Bam H1 is from Bacillus
amyloliquefaciens, strain H
The ‘Toolkit’
Restriction enzymes that generate ‘sticky ends’
are very useful tools to the genetic engineer
The same restriction enzyme
recognises the same nucleotide
sequence in the DNA from
different species and creates
the same ‘sticky ends’
When the DNA fragments from the
two different species are mixed
together, the complementary bases of
their ‘sticky ends’ will be attracted to
one another and form hydrogen bonds
In this way, DNA fragments from
different sources can be brought
together and joined
DNA ligase is the enzyme that
seals fragments of DNA together
Fragment of DNA from species Y
Fragment of DNA from species X
G
C T
A A
T
Complementary bases on the
sticky ends of the DNA from
the different species are
attracted to one another
T
C
G
A A
Complementary sticky ends
created by cutting the DNA
from each species with the
same restriction enzyme
T
C
A A
G
G A A
T
C
C T
A A G
G
A A
C T
Hydrogen bonds
form between the
bases and the
enzyme DNA ligase
seals the sugarphosphate backbone
of the DNA molecule
T
T
T
T
T
Recombinant DNA is formed
The ‘Toolkit’
VECTORS
Vectors are carrier DNA molecules into which DNA fragments containing
specific genes can be inserted
Vectors are the means by which selected genes are carried into host cells
where the desired gene is then cloned
The isolated plasmids of bacterial cells and the DNA of bacteriophages (viruses
that infect bacteria) are frequently used as vectors
Plasmids are small, circular, self-replicating double-stranded DNA molecules
found in bacterial cells,which are separate from the main bacterial chromosome
main
chromosome
plasmid
The ‘Toolkit’
Genes coding for ‘desirable products’ can be spliced into plasmids to form
RECOMBINANT PLASMIDS
When these plasmids are taken up by bacterial host cells, they replicate along
with the host cell and clone the desired gene
Plasmids are obtained from cultures of bacterial cells; bacterial cells are
broken open and the plasmids are separated out by centrifugation
Homogenised bacterial cells, when subjected to centrifugation, provide the
plasmids into which foreign genes can be inserted
Making Recombinant Plasmids
Total human DNA extracted from human cells
- known as genomic DNA
Plasmid
DNA fragment with sticky ends complementary
to those on the ‘cut’ plasmid
Both the plasmid and the human DNA are treated
with the SAME restriction enzyme so that the DNA from
both sources will have complementary ‘sticky ends’
The two DNA molecules
are attracted to one another
and, in the presence of DNA
ligase, form a recombinant
Recombinant plasmid
DNA molecule
When host bacterial cells are mixed with these recombinant plasmids, they may take them
up and become transformed; these bacterial cells are now described as transgenic
organisms as they contain and express the genetic material from a different species
When this transformed
bacterial cell divides, the
recombinant plasmid
replicates and copies of the
plasmid (containing the
foreign DNA) are passed to
the daughter cells
The foreign DNA has been cloned