I Recombinant DNA technology
? 1 Restriction enzymes
? 2 Nucleic acid hybridization
? 3 DNA cloning
? 4 Viruses
? 5 DNA sequencing
? 6 Polymerase chain reaction
? Overview
? Restriction enzyme digestion
? Nomenclature
? Gel electrophoresis
? Restriction maps
? Restriction fragment length
polymorphisms(RFLP)
Restriction enzymes
Overview
? Restriction enzymes allow DNA to be cut at
specific sites; Nucleic acid hybridization
allows the detection of specific nucleic
acid sequences; DNA sequencing can be
used to easily determine the nucleotide
sequence of a DNA molecule,
Restriction endonuclease
(Restriction enzyme)
? Bacterial enzymes which cut DNA into
defined and reproducible fragments
? Identified in the late 1960s
? Key discovery which allowed the DNA
cloning to become a reality
Restriction endonuclease
(origination)
? One component of the bacterial restriction-modification
system,a natural defense mechanism of bacteria to against
the introduction of foreign DNA into the cell
? Restriction endonuclease,recognize a short,symmetrical
DNA sequence,and cut DNA backbone in each strand at a
specific site within that sequence (kill foreign DNA)
? Mythylase,methylates C or A of the cellular DNA
Types of Restriction
endonuclease
Type I Type II Type III
Functions Endonuclease &
methylase
Endonuclease Endonuclease
Conditions ATP,Mb2+ Mg2+ ATP,Mg2+
Recognition
sequences
EcoK,AACN6GTGC
EcoB,TGAN8TGCT
Palindromic(回文序列） EcoP1,AGACC
EcoP15,CAGCAG
Cutting sites At least 1000bp away At or close to recog,
seq
24-26 bp away
Restriction enzymes
Recognize 4-8 bp palindromic sequences,Most
commonly used enzymes recognize 6 bp which
occurs at a rate of 46=4096 bp,(44=256 bp;
48=65536 bp)
1,Highly specific
2,Commercially available
3,Require Mg2+ for enzymatic activity
4,Compatible ends from different enzymes,
5’ GAATTC 3’
3’ CTTAAG 5’ e.g,EcoRI site,
Recognition sequences
5’ protruding ends 3’ protruding ends
5’-CCCGGG-3’
3’-GGGCCC-5’
5’-CCC-OH
3’-GGG- p
p -GGG-3’
OH-CCC-5’ +
SmaI
blunt ends
Cohesive/sticky ends
Restriction sequences
Restriction digestion
Agarose,a polysaccharide derived from seaweed,
which forms a solid gel when dissolved in aqueous
solution (0.5%-2%)
- ve electrode + ve electrode
Negatively charged DNA
Agarose gel electrophoresis
supercoiled
nicked
Agarose gel electrophoresis
Covalently join the DNA molecules
with the base-pairing cohesive
ends,or blunt ends,if the 5’-ends
have phosphate groups,
DNA ligation
X
if the vector is phosphorylated
Recombinant DNA molecules
Fig
? The hybridization reaction
? Monitoring specific nucleic acid
sequences
? Southern blotting
? Northern blotting
? In situ hybridization
Nucleic acid hybridization
The hybridization reaction
? Double-stranded DNA denatures into single
strands as the temperature rises but renatures
into a double-stranded structure as the
temperature falls, Any two single-stranded
nucleic acid molecules can form double-
stranded structures (hybridize ) provided that
have sufficient complementary nucleotide
sequence to make the resulting hybrid stable
under the reaction conditions,
Monitoring specific nucleic acid
sequences
? The concentration of specific nucleic acid
sequence in a sample can be measured by
hybridization with a suitable labeled DNA
probe, After hybridization,nuclear is used
to destroy unhybridized probe and the probe
remaining is a measure of the concentration
of the target sequence,
Southern biotting
? Southern blotting involves electrophoresis
of DNA molecules in an agarose gel and
then blotting the separated DNA bands on
to a nitrocellulose filter,The filter is then
incubated with a labeled DNA probe to
detect those seq, parated DNA bands that
contain sequences complementary to the
probe,
Northern blotting
? Northern blotting is analogous to
Southern blotting except that the
sample nucleic acid that is separated
by gel electrophoresis is RNA rather
than DNA 。
In situ hybridization
? For in situ hybridization,a tissue sample
is incubated with a labeled nucleic acid
probe,excess probe is washed away and
the location of hybridized probe is
examined,The technique enables the
spatial localization of gene expression to
be determined as well as the location of
individual genes on chromosomes,
? The principle of DNA cloning
? The basics of DNA cloning
? DNA libraries
? Screening DNA libraries
DNA cloning
Basic procedure of DNA cloning
DNA libraries
? DNA libraries are sets of DNA clones,each of which
has been derived from the insertion of a different
fragment into a vector followed by propagation in the
host,
? A clone is a genetically distinct individual or set of
identical individuals
Genomic DNA libraries CDNA libraries
Genomic libraries
prepared form random fragments of
genomic DNA,which may be
inefficient to find a gene because of
the huge abundance of the non-
coding DNA
cDNA libraries
DNA copies (cDNA) synthesized from
the mRNA by reverse transcription are
inserted into a vector to form a cDNA
library,Much more efficient in
identifying a gene,but do not contain
DNA coding for functional RNA or
noncoding sequence,
? Overview
? Bacteriophages
? Animal viruses
virus
Overview
? A virus particle (virion) has a DNA or
RNA genome packaged inside a protein
capsid,
Each virus can replicate only by infecting
a limited range of host cells,Viruses exit
the host cell by budding through the
plasma membrane without causing cell
death,
Bacteriophages
? Bacteriophages adsorb to a bacterial cell
surface and inject the phage DNA
through the cell wall into the cytosol,In
the lytic cycle,this DNA then replicates
inside the cell and is packaged within
newly synthesized capsids,eventually
being released by cell lysis,
Animal viruses
? Permissive cells infected with an animal
DNA virus enter a lytic cycle,but in
nonpermissive cells an animal virus may
become integrated into the
nucleargenome or become a plasmid.In
this case the virus is known as a DNA
tumor virus,
? Two methods for DNA sequencing
? Chain termination method
? Automated DNA sequencing
DNA sequencing
Two methods for DNA sequencing
? DNA can be sequenced by the chemical
method or the chain termination
procedure,The latter is now the method of
choice in which the (single-stranded) DNA
to be sequenced serves as the template
for the synthesis of a complementary
strand when supplied with a specific
primer and E.coli DNA polymeraseⅠ,
Chain termination method
? Four incubation mixtures are set
up,each containing the DNA template,a
specific DNA primer,E.coli DNA
polymerase Ⅰ and all four
deoxyribonucleoside triphosphates
(dNTPs),In addition,each mixture
contains a different dideoxynucleoside
triphosphate analog,ddATP,ddCTP
ddGTP or ddTTP,
Automated DNA sequencing
? Automated DNA sequencing uses the chain
termination method but with an
oligonucleotide primer labeled with a
fluorescent dye,The order in which the
different fluorescently labeled termination
products elute from the gel gives the DNA
sequence,
? Principles of PCR Applications
of PCR
Polymerase ChainReaction
Applications of PCR
? PCR has made a huge impact in
molecular biology,with many
applications in areas such as cloning,
sequencing,the creation of specific
mutations,medical diagnosis and
forensic medicine,
Principles of PCR
? The polymerase chain reaction(PCR)
allows an extremely large number of
copies to be synthesized of any given
DNA sequence,which consists of three
steps,denaturation,primer annealing
and elongation,
? 1 Restriction enzymes
? 2 Nucleic acid hybridization
? 3 DNA cloning
? 4 Viruses
? 5 DNA sequencing
? 6 Polymerase chain reaction
? Overview
? Restriction enzyme digestion
? Nomenclature
? Gel electrophoresis
? Restriction maps
? Restriction fragment length
polymorphisms(RFLP)
Restriction enzymes
Overview
? Restriction enzymes allow DNA to be cut at
specific sites; Nucleic acid hybridization
allows the detection of specific nucleic
acid sequences; DNA sequencing can be
used to easily determine the nucleotide
sequence of a DNA molecule,
Restriction endonuclease
(Restriction enzyme)
? Bacterial enzymes which cut DNA into
defined and reproducible fragments
? Identified in the late 1960s
? Key discovery which allowed the DNA
cloning to become a reality
Restriction endonuclease
(origination)
? One component of the bacterial restriction-modification
system,a natural defense mechanism of bacteria to against
the introduction of foreign DNA into the cell
? Restriction endonuclease,recognize a short,symmetrical
DNA sequence,and cut DNA backbone in each strand at a
specific site within that sequence (kill foreign DNA)
? Mythylase,methylates C or A of the cellular DNA
Types of Restriction
endonuclease
Type I Type II Type III
Functions Endonuclease &
methylase
Endonuclease Endonuclease
Conditions ATP,Mb2+ Mg2+ ATP,Mg2+
Recognition
sequences
EcoK,AACN6GTGC
EcoB,TGAN8TGCT
Palindromic(回文序列） EcoP1,AGACC
EcoP15,CAGCAG
Cutting sites At least 1000bp away At or close to recog,
seq
24-26 bp away
Restriction enzymes
Recognize 4-8 bp palindromic sequences,Most
commonly used enzymes recognize 6 bp which
occurs at a rate of 46=4096 bp,(44=256 bp;
48=65536 bp)
1,Highly specific
2,Commercially available
3,Require Mg2+ for enzymatic activity
4,Compatible ends from different enzymes,
5’ GAATTC 3’
3’ CTTAAG 5’ e.g,EcoRI site,
Recognition sequences
5’ protruding ends 3’ protruding ends
5’-CCCGGG-3’
3’-GGGCCC-5’
5’-CCC-OH
3’-GGG- p
p -GGG-3’
OH-CCC-5’ +
SmaI
blunt ends
Cohesive/sticky ends
Restriction sequences
Restriction digestion
Agarose,a polysaccharide derived from seaweed,
which forms a solid gel when dissolved in aqueous
solution (0.5%-2%)
- ve electrode + ve electrode
Negatively charged DNA
Agarose gel electrophoresis
supercoiled
nicked
Agarose gel electrophoresis
Covalently join the DNA molecules
with the base-pairing cohesive
ends,or blunt ends,if the 5’-ends
have phosphate groups,
DNA ligation
X
if the vector is phosphorylated
Recombinant DNA molecules
Fig
? The hybridization reaction
? Monitoring specific nucleic acid
sequences
? Southern blotting
? Northern blotting
? In situ hybridization
Nucleic acid hybridization
The hybridization reaction
? Double-stranded DNA denatures into single
strands as the temperature rises but renatures
into a double-stranded structure as the
temperature falls, Any two single-stranded
nucleic acid molecules can form double-
stranded structures (hybridize ) provided that
have sufficient complementary nucleotide
sequence to make the resulting hybrid stable
under the reaction conditions,
Monitoring specific nucleic acid
sequences
? The concentration of specific nucleic acid
sequence in a sample can be measured by
hybridization with a suitable labeled DNA
probe, After hybridization,nuclear is used
to destroy unhybridized probe and the probe
remaining is a measure of the concentration
of the target sequence,
Southern biotting
? Southern blotting involves electrophoresis
of DNA molecules in an agarose gel and
then blotting the separated DNA bands on
to a nitrocellulose filter,The filter is then
incubated with a labeled DNA probe to
detect those seq, parated DNA bands that
contain sequences complementary to the
probe,
Northern blotting
? Northern blotting is analogous to
Southern blotting except that the
sample nucleic acid that is separated
by gel electrophoresis is RNA rather
than DNA 。
In situ hybridization
? For in situ hybridization,a tissue sample
is incubated with a labeled nucleic acid
probe,excess probe is washed away and
the location of hybridized probe is
examined,The technique enables the
spatial localization of gene expression to
be determined as well as the location of
individual genes on chromosomes,
? The principle of DNA cloning
? The basics of DNA cloning
? DNA libraries
? Screening DNA libraries
DNA cloning
Basic procedure of DNA cloning
DNA libraries
? DNA libraries are sets of DNA clones,each of which
has been derived from the insertion of a different
fragment into a vector followed by propagation in the
host,
? A clone is a genetically distinct individual or set of
identical individuals
Genomic DNA libraries CDNA libraries
Genomic libraries
prepared form random fragments of
genomic DNA,which may be
inefficient to find a gene because of
the huge abundance of the non-
coding DNA
cDNA libraries
DNA copies (cDNA) synthesized from
the mRNA by reverse transcription are
inserted into a vector to form a cDNA
library,Much more efficient in
identifying a gene,but do not contain
DNA coding for functional RNA or
noncoding sequence,
? Overview
? Bacteriophages
? Animal viruses
virus
Overview
? A virus particle (virion) has a DNA or
RNA genome packaged inside a protein
capsid,
Each virus can replicate only by infecting
a limited range of host cells,Viruses exit
the host cell by budding through the
plasma membrane without causing cell
death,
Bacteriophages
? Bacteriophages adsorb to a bacterial cell
surface and inject the phage DNA
through the cell wall into the cytosol,In
the lytic cycle,this DNA then replicates
inside the cell and is packaged within
newly synthesized capsids,eventually
being released by cell lysis,
Animal viruses
? Permissive cells infected with an animal
DNA virus enter a lytic cycle,but in
nonpermissive cells an animal virus may
become integrated into the
nucleargenome or become a plasmid.In
this case the virus is known as a DNA
tumor virus,
? Two methods for DNA sequencing
? Chain termination method
? Automated DNA sequencing
DNA sequencing
Two methods for DNA sequencing
? DNA can be sequenced by the chemical
method or the chain termination
procedure,The latter is now the method of
choice in which the (single-stranded) DNA
to be sequenced serves as the template
for the synthesis of a complementary
strand when supplied with a specific
primer and E.coli DNA polymeraseⅠ,
Chain termination method
? Four incubation mixtures are set
up,each containing the DNA template,a
specific DNA primer,E.coli DNA
polymerase Ⅰ and all four
deoxyribonucleoside triphosphates
(dNTPs),In addition,each mixture
contains a different dideoxynucleoside
triphosphate analog,ddATP,ddCTP
ddGTP or ddTTP,
Automated DNA sequencing
? Automated DNA sequencing uses the chain
termination method but with an
oligonucleotide primer labeled with a
fluorescent dye,The order in which the
different fluorescently labeled termination
products elute from the gel gives the DNA
sequence,
? Principles of PCR Applications
of PCR
Polymerase ChainReaction
Applications of PCR
? PCR has made a huge impact in
molecular biology,with many
applications in areas such as cloning,
sequencing,the creation of specific
mutations,medical diagnosis and
forensic medicine,
Principles of PCR
? The polymerase chain reaction(PCR)
allows an extremely large number of
copies to be synthesized of any given
DNA sequence,which consists of three
steps,denaturation,primer annealing
and elongation,
