Download Recombinant DNA (DNA Cloning)

Recombinant DNA (DNA Cloning)
SC.912.L.16.12
What is DNA cloning?
• When DNA is
extracted from an
organism, all its
genes are obtained
• In gene (DNA)
cloning a particular
gene is copied
(cloned)
Whole organisms are cloned too,
but differently
DNA cloning and Recombinant DNA
• Major tool-restriction enzymes
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discovered in late 1960’s
cuts DNA at restriction site
highly specific
manufactured by bacteria
• Cloning vector - carrier for moving DNA into a cell;
such as a bacterial virus or plasmid into which foreign
DNA can be inserted
• Recombinant DNA: joining together of two fragments
of DNA that are not normally joined together (e.g.
joining together of eukaryotic DNA and prokaryotic
DNA - usually in a cloning vector)
Why Clone DNA?
• A particular gene can be isolated and its
nucleotide sequence determined
• Control sequences of DNA can be identified &
analyzed
• Protein/enzyme/RNA function can be
investigated
• Mutations can be identified, e.g. gene defects
related to specific diseases
• Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production, insect
resistance, etc.
How is DNA cloned?
Blood sample
• DNA is extractedhere from blood
• Restriction enzymes,
e.g. EcoRI, HindIII,
etc., cut the DNA into
small pieces
• Different DNA pieces
cut with the same
enzyme can join, or
recombine.
DNA
Restriction enzymes
Restriction Enzymes
• Bacteria have learned to "restrict" the possibility of attack
from foreign DNA by means of "restriction enzymes”.
• Cut up “foreign” DNA that invades the cell.
• Type II and III restriction enzymes cleave DNA chains at
selected sites.
• Enzymes may recognize 4, 6 or more bases in selecting
sites for cleavage.
Basics of type II Restriction Enzymes
• No ATP requirement.
• Recognition sites in double stranded DNA have a 2-fold axis
of symmetry – a “palindrome”.
• Cleavage can leave staggered or "sticky" ends or can produce
"blunt” ends.
The action of a restriction enzyme, EcoRI
Note: EcoRI gives a ‘sticky’ end
Cut and Paste DNA fragments with complementary ends (2 sticky
ends)
DNA Cloning, II
• Bacterial plasmids
(small circular DNA
additional to a
bacteria’s regular DNA)
are cut with the same
restriction enzyme
• A chunk of DNA can
thus be inserted into the
plasmid DNA to form a
“recombinant”
DNA cloning, III
• The recombinant
plasmids are then
mixed with bacteria
which have been
treated to make them
“competent”, or
capable of taking in
the plasmids
• This insertion is called
transformation
DNA Cloning IV
• The plasmids have
naturally occurring
genes for antibiotic
resistance
• Bacteria containing
plasmids with these
genes will grow on a
medium containing the
antibiotic- the others
die, so only transformed
bacteria survive
PCR
polymerase chain reaction
• DNA doubled in each cycle
– starting with 1 molecule of DNA
– 1 million copies after 20 cycles
• Key to procedure is thermostable DNA
polymerase (Taq polymerase)
• Ability to amplify minute amounts of DNA
valuable in many disciplines
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basic and applied research
criminal forensic science
ecology
analysis of ancient DNA