04/27/2017
Genetic engineering
Genetic engineering
• Objectives;
– the students will learn basic techniques of genetic
engineering– those used in cloning, altering of
genes, and express them efficiently in host
organisms
• Performing genetics only in vivo (in living
organisms) has many limitations that can be
overcome by manipulating DNA in vitro (in a
test tube).
1
, 04/27/2017
Methods of manipulating DNA
• Genetic engineering refers to the use of in vitro techniques
to alter genetic material in the laboratory.
• Such altered genetic material may be reinserted into the
original source organism or into some other host organism.
• Genetic engineering depends upon our ability to cut DNA
into specific fragments and to purify these for further
manipulation.
• Basic tools of genetic engineering
– Restriction enzymes,
– the separation of nucleic acids by electrophoresis,
– nucleic acid hybridization, and
– molecular cloning.
Restriction and modification enzymes
Restriction enzymes
• All cells contain enzymes that can chemically modify DNA in
one way or another. One major class of such enzymes is the
restriction endonucleases, or restriction enzymes for short.
• Restriction enzymes recognize specific base sequences (recognition
sequences) within DNA and cut the DNA.
• Although they are widespread among prokaryotes (both Bacteria
and Archaea), they are very rare in eukaryotes.
• In vivo restriction enzymes protect prokaryotes from hostile foreign
DNA such as virus genomes.
• However, restriction enzymes are also essential for in vitro DNA
manipulation, and their discovery gave birth to the
field of genetic engineering.
2
, 04/27/2017
• There are three types of restriction enzymes
– Type I, II, III
• Type I and III restriction enzymes bind to the DNA at
their recognition sequences but cut the DNA a
considerable distance away.
• However, type II restriction enzymes cleave the DNA
within their recognition sequences, making this class of
enzymes much more useful for the specific
manipulation of DNA
• Restriction enzyme EcoRI
• The sequence of DNA
recognized by the restriction
endonuclease EcoRI. The red
arrows indicate the bonds
cleaved by the enzyme. The
dashed line indicates the axis of
symmetry of the sequence.
• Note the appearance of DNA
after cutting with restriction
enzyme EcoRI - the
singlestranded “sticky ends.”
3
Genetic engineering
Genetic engineering
• Objectives;
– the students will learn basic techniques of genetic
engineering– those used in cloning, altering of
genes, and express them efficiently in host
organisms
• Performing genetics only in vivo (in living
organisms) has many limitations that can be
overcome by manipulating DNA in vitro (in a
test tube).
1
, 04/27/2017
Methods of manipulating DNA
• Genetic engineering refers to the use of in vitro techniques
to alter genetic material in the laboratory.
• Such altered genetic material may be reinserted into the
original source organism or into some other host organism.
• Genetic engineering depends upon our ability to cut DNA
into specific fragments and to purify these for further
manipulation.
• Basic tools of genetic engineering
– Restriction enzymes,
– the separation of nucleic acids by electrophoresis,
– nucleic acid hybridization, and
– molecular cloning.
Restriction and modification enzymes
Restriction enzymes
• All cells contain enzymes that can chemically modify DNA in
one way or another. One major class of such enzymes is the
restriction endonucleases, or restriction enzymes for short.
• Restriction enzymes recognize specific base sequences (recognition
sequences) within DNA and cut the DNA.
• Although they are widespread among prokaryotes (both Bacteria
and Archaea), they are very rare in eukaryotes.
• In vivo restriction enzymes protect prokaryotes from hostile foreign
DNA such as virus genomes.
• However, restriction enzymes are also essential for in vitro DNA
manipulation, and their discovery gave birth to the
field of genetic engineering.
2
, 04/27/2017
• There are three types of restriction enzymes
– Type I, II, III
• Type I and III restriction enzymes bind to the DNA at
their recognition sequences but cut the DNA a
considerable distance away.
• However, type II restriction enzymes cleave the DNA
within their recognition sequences, making this class of
enzymes much more useful for the specific
manipulation of DNA
• Restriction enzyme EcoRI
• The sequence of DNA
recognized by the restriction
endonuclease EcoRI. The red
arrows indicate the bonds
cleaved by the enzyme. The
dashed line indicates the axis of
symmetry of the sequence.
• Note the appearance of DNA
after cutting with restriction
enzyme EcoRI - the
singlestranded “sticky ends.”
3
