Gene Manipulation
Medical Genetics
Recombination DNA Technique
Techniques for joining DNA molecules
in vitro and introducing them into living
cells where they replicate.
Insert specific DNA into vector
Clone in host cells
Cloning
Isolate cloned DNA
foreign DNA bacterial cell
Schematic of
recombination DNA technique
vector
restriction
endonuclease
foreign DNA bacterial cell
Schematic of
recombination DNA technique
ligase
vector
restriction
endonuclease
foreign DNA bacterial cell
host
Schematic of
recombination DNA technique
ligase
vector
restriction
endonuclease
foreign DNA bacterial cell
host
Schematic of
recombination DNA technique
Schematic of
recombination DNA technique
Cloning
Amp Amp＋ X-gal
Schematic of
recombination DNA technique
“Blue-white” screening
Recombination DNA
DNA clone
cDNA clone
Artificial gene clone
PCR product clone
Gene Transfer
Transgenic animal
Transgenic mouse
Embryonic stem cell,ES cell
Gene Transfer
Transgenic animal
Transgenic mouse
Embryonic stem cell,ES cell
Gene knockout mouse
Gene Transfer
Somatic cell cloning
Transgenic animal
Somatic Cell Cloning
Molecular Hybridization
DNA library
Nucleic acid probe
Method
DNA Library
Genomic DNA library
Chromosome specific library
cDNA library
Nucleic Acid Probe
A labelled segment of DNA (RNA) that
is able,after a DNA hybridization reaction,
to detect a specific DNA sequence in a
mixture of sequences.
Type of Probe
DNA probe
RNA probe
Oligonucleotide probe
Degenerate probe
Unique EST-base probe
Degenerate Probe
Leu Cys Ile Tyr Met His Gln Asp Ser
CT
T
C
A
G
TG TC AT
T
C
A TA
T
C CA
T
CATG CA
A
G GA
T
C TC
T
C
A
G
TT AG AGTC
Type of Probe
DNA probe
RNA probe
Oligonucleotide probe
Degenerate probe
Unique EST-base probe
Probe Labelling
Nick translation
Random-primed labeling
PCR amplification
End labelling
Denaturation or Melting
Renaturation or Annealing
Homoduplexes & Heteroduplexes
Molecular Hybridization
DNA hybridization
Southern blot
Molecular Hybridization
Southern Blot
-
+
Southern Blot
Pathologic mutation
Pathologic Mutation
Sickle cell anemia (HbS),code 5~7
A?A?A?S?S?S
MstⅡ,CCTNAGG
1.2 Kb 0.2Kb
1.40 Kb
1.20 Kb
0.20 Kb
A,CCTGAGGAG
S,CCTGTGGAG
Southern Blot
Pathologic mutation
Restriction site polymorphism,RSP
DNA finger printing
DNA Finger Printing
VNTR (variable number of tandom repeats)
Southern Blot
Pathologic mutation
Restriction site polymorphism,RSP
DNA finger printing
STR (short terminal repeat)
Gene deletion
DNA hybridization
Southern blot
Molecular Hybridization
Dot and Slot hybridization
Allele-specific oligonucleotide,ASO
ASO Hybridization
His Leu Thr Pro Glu Glu Lys Ser Ala
CAT CTG ACT CCT GAG GAG AAG TCT GCC
His Leu Thr Pro Val Glu Lys Ser Ala
CAT CTG ACT CCT GTG GAG AAG TCT GCC
CTG ACT CCT GAG GAG AAG TCT
CTG ACT CCT GTG GAG AAG TCT
DNA hybridization
Molecular Hybridization
RNA hybridization
Northern blot
DNA hybridization
Molecular Hybridization
RNA hybridization
Protein hybridization
Western blot (immunoblotting)
Immunohistochemistry
(immunocytochemistry)
Polymerase Chain Reaction
It’s a means of selectively amplifying a
particular segment of DNA in vitro.
The basic principle of replicating a piece of
DNA using two primers had already been
described by Gobind Khorana in 1971.
Polymerase Chain Reaction
Mullis F
The Nobel Prize in Chemistry 1993
"for his invention of the
polymerase chain reaction
(PCR) method"
Polymerase Chain Reaction
Denaturing step,94~95℃
Denatures the double stranded DNA into
single strands
95℃
72℃
55℃
Polymerase Chain Reaction
Annealing step,45~60℃
Denaturing step,94~95℃
Allows the primers to attach to complementary
strand of DNA
95℃
72℃
55℃
Polymerase Chain Reaction
Extension step,72℃
Annealing step,45~60℃
Denaturing step,94~95℃
Optimal temperature for Taq polymerase to attach
to and extend the new strand of DNA
95℃
72℃
55℃
Polymerase Chain Reaction
It can be thought of as a molecular
photocopier.
Polymerase Chain Reaction
Advantages
Speed
Sensitive
Stable
Disadvantages
Need sequence information
Limited amplification size
Infidelity of DNA replication
Restriction Endonuclease
Any one of many enzymes that
cleave foreign DNA molecules at
specific recognition sites.
TypeⅠ
TypeⅡ
TypeⅢ
Blunt end & Sticky end
Restriction Endonuclease
HaeⅢ 5’ 3’3’ 5’G G C CC C G G 5’ 3’3’ 5’G GC C C CG G
MboⅠ 5’ 3’3’ 5’G A T CC T A G 5’ 3’3’ 5’G A T CC T A G
BamHⅠ 5’ 3’3’ 5’G G A T C CC C T A G G 5’ 3’3’ 5’GC C T A GG A T C CG
PstⅠ 5’ 3’3’ 5’C T G C A GG A C G T C 5’ 3’3’ 5’C T G C AG GA C G T C
Back
DNA Ligase
Enzymes that catalyze the formation of
a phosphodiester bond between adjacent
3’-OH and 5’-P termini in DNA.
Vector
A self-replicating DNA molecule that
transfers a DNA segment between host
cells.
Small
A cloning site (multi-clone site)
A replicator (origin of replication)
A selectable marker
Plasmid
An extrachromosomal genetic element
found in a variety of bacterial species.
Plasmid are double-stranded,closed DNA
molecules.
Plasmid
agtgaattCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGcgtaatcatggtcat
pUC19
2686 bp Ori
ApR
LacZ
EcoRⅠ
SacⅠ
KpnⅠ
SmaⅠ XbaⅠ
BamHⅠ SalⅠ SphⅠ
PstⅠ HindⅢ
Lambda Phage
A double-stranded DNA virus that is
engineered to serve as a receptor for
foreign DNA fragments in recombinant
DNA experiments.
Plasmid
Cos site
Lytic & Lysogenic
Replacement λ vector
Insertion λ vector
Cosmid
Vectors designed for cloned large
fragment of eukaryotic DNA,The term
signifies that the vector is a plasmid into
which phage lambda cos sites have been
inserted.
Cosmid
pJB8
5.4 kb
Sal Ⅰ
Others
P1 phage & PAC
BAC,bacterial artificial chromosome
YAC,yeast artificial chromosome
Centromere
Telomere
ARS,autonomous replication sequence
Back
Vector
Vector Targeting Fragment Length
Plasmid 0~10 kb
Replacement λ vector 0~10 kb
Insertion λ vector 9~23 kb
Cosmid 33~44 kb
P1 phage 70~100 kb
PAC 130~150 kb
BAC 300 kb
YAC 0.2~2.0 Mb
Nick Translation
p A p C p G p T p T p A p C p G p G p A p C p T p G p T p T p A p C p C p G p A p T
p T p G p C p A p A p T p G p C p C p T p G p A p C p A p A p T p G p G p C p T p A
DNaseⅠ DNA PolymeraseⅠ
dATP
dCTP
dGTP
dTTP
Nick Translation
p A p C p G p T p T p A C p G p G p A p C p T p G p T p T p A p C p C p G p A p T
p T p G p C p A p A p T p G p C p C p T p G p A p C p A p A p T p G p G p C p T p A
DNaseⅠ
dATP
dCTP
dGTP
dTTP
Nick Translation
p A p C p G p T p T p A C p G p G p A p C p T p G p T p T p A p C p C p G p A p T
p T p G p C p A p A p T p G p C p C p T p G p A p C p A p A p T p G p G p C p T p A
OH PDNA PolymeraseⅠ
dATP
dCTP
dGTP
dTTP
Nick Translation
p A p C p G p T p T p A p G p G p A p C p T p G p T p T p A p C p C p G p A p T
p T p G p C p A p A p T p G p C p C p T p G p A p C p A p A p T p G p G p C p T p A
dCMP
dATP
dCTP
dGTP
dTTP DNA
PolymeraseⅠ
p A p C p G p T p T p A p G p G p A p C p T p G p T p T p A p C p C p G p A p T
p T p G p C p A p A p T p G p C p C p T p G p A p C p A p A p T p G p G p C p T p A
pC
dATP
dCTP
dGTP
dTTP DNA
PolymeraseⅠ
Nick Translation
Back
Nick Translation
p A p C p G p T p T p A p C p G p G p A p C p T p G p T p T p A p C p C p G p A p T
p T p G p C p A p A p T p G p C p C p T p G p A p C p A p A p T p G p G p C p T p A
dATP
dCTP
dGTP
dTTP
DNA PolymeraseⅠ
Random-Primed Labeling
5’ 3’
5’3’
dATP dCTP
dGTP dTTP
Klenow
Klenow Klenow
Back
Random-Primed Labeling
5’ 3’
5’3’
Medical Genetics
Recombination DNA Technique
Techniques for joining DNA molecules
in vitro and introducing them into living
cells where they replicate.
Insert specific DNA into vector
Clone in host cells
Cloning
Isolate cloned DNA
foreign DNA bacterial cell
Schematic of
recombination DNA technique
vector
restriction
endonuclease
foreign DNA bacterial cell
Schematic of
recombination DNA technique
ligase
vector
restriction
endonuclease
foreign DNA bacterial cell
host
Schematic of
recombination DNA technique
ligase
vector
restriction
endonuclease
foreign DNA bacterial cell
host
Schematic of
recombination DNA technique
Schematic of
recombination DNA technique
Cloning
Amp Amp＋ X-gal
Schematic of
recombination DNA technique
“Blue-white” screening
Recombination DNA
DNA clone
cDNA clone
Artificial gene clone
PCR product clone
Gene Transfer
Transgenic animal
Transgenic mouse
Embryonic stem cell,ES cell
Gene Transfer
Transgenic animal
Transgenic mouse
Embryonic stem cell,ES cell
Gene knockout mouse
Gene Transfer
Somatic cell cloning
Transgenic animal
Somatic Cell Cloning
Molecular Hybridization
DNA library
Nucleic acid probe
Method
DNA Library
Genomic DNA library
Chromosome specific library
cDNA library
Nucleic Acid Probe
A labelled segment of DNA (RNA) that
is able,after a DNA hybridization reaction,
to detect a specific DNA sequence in a
mixture of sequences.
Type of Probe
DNA probe
RNA probe
Oligonucleotide probe
Degenerate probe
Unique EST-base probe
Degenerate Probe
Leu Cys Ile Tyr Met His Gln Asp Ser
CT
T
C
A
G
TG TC AT
T
C
A TA
T
C CA
T
CATG CA
A
G GA
T
C TC
T
C
A
G
TT AG AGTC
Type of Probe
DNA probe
RNA probe
Oligonucleotide probe
Degenerate probe
Unique EST-base probe
Probe Labelling
Nick translation
Random-primed labeling
PCR amplification
End labelling
Denaturation or Melting
Renaturation or Annealing
Homoduplexes & Heteroduplexes
Molecular Hybridization
DNA hybridization
Southern blot
Molecular Hybridization
Southern Blot
-
+
Southern Blot
Pathologic mutation
Pathologic Mutation
Sickle cell anemia (HbS),code 5~7
A?A?A?S?S?S
MstⅡ,CCTNAGG
1.2 Kb 0.2Kb
1.40 Kb
1.20 Kb
0.20 Kb
A,CCTGAGGAG
S,CCTGTGGAG
Southern Blot
Pathologic mutation
Restriction site polymorphism,RSP
DNA finger printing
DNA Finger Printing
VNTR (variable number of tandom repeats)
Southern Blot
Pathologic mutation
Restriction site polymorphism,RSP
DNA finger printing
STR (short terminal repeat)
Gene deletion
DNA hybridization
Southern blot
Molecular Hybridization
Dot and Slot hybridization
Allele-specific oligonucleotide,ASO
ASO Hybridization
His Leu Thr Pro Glu Glu Lys Ser Ala
CAT CTG ACT CCT GAG GAG AAG TCT GCC
His Leu Thr Pro Val Glu Lys Ser Ala
CAT CTG ACT CCT GTG GAG AAG TCT GCC
CTG ACT CCT GAG GAG AAG TCT
CTG ACT CCT GTG GAG AAG TCT
DNA hybridization
Molecular Hybridization
RNA hybridization
Northern blot
DNA hybridization
Molecular Hybridization
RNA hybridization
Protein hybridization
Western blot (immunoblotting)
Immunohistochemistry
(immunocytochemistry)
Polymerase Chain Reaction
It’s a means of selectively amplifying a
particular segment of DNA in vitro.
The basic principle of replicating a piece of
DNA using two primers had already been
described by Gobind Khorana in 1971.
Polymerase Chain Reaction
Mullis F
The Nobel Prize in Chemistry 1993
"for his invention of the
polymerase chain reaction
(PCR) method"
Polymerase Chain Reaction
Denaturing step,94~95℃
Denatures the double stranded DNA into
single strands
95℃
72℃
55℃
Polymerase Chain Reaction
Annealing step,45~60℃
Denaturing step,94~95℃
Allows the primers to attach to complementary
strand of DNA
95℃
72℃
55℃
Polymerase Chain Reaction
Extension step,72℃
Annealing step,45~60℃
Denaturing step,94~95℃
Optimal temperature for Taq polymerase to attach
to and extend the new strand of DNA
95℃
72℃
55℃
Polymerase Chain Reaction
It can be thought of as a molecular
photocopier.
Polymerase Chain Reaction
Advantages
Speed
Sensitive
Stable
Disadvantages
Need sequence information
Limited amplification size
Infidelity of DNA replication
Restriction Endonuclease
Any one of many enzymes that
cleave foreign DNA molecules at
specific recognition sites.
TypeⅠ
TypeⅡ
TypeⅢ
Blunt end & Sticky end
Restriction Endonuclease
HaeⅢ 5’ 3’3’ 5’G G C CC C G G 5’ 3’3’ 5’G GC C C CG G
MboⅠ 5’ 3’3’ 5’G A T CC T A G 5’ 3’3’ 5’G A T CC T A G
BamHⅠ 5’ 3’3’ 5’G G A T C CC C T A G G 5’ 3’3’ 5’GC C T A GG A T C CG
PstⅠ 5’ 3’3’ 5’C T G C A GG A C G T C 5’ 3’3’ 5’C T G C AG GA C G T C
Back
DNA Ligase
Enzymes that catalyze the formation of
a phosphodiester bond between adjacent
3’-OH and 5’-P termini in DNA.
Vector
A self-replicating DNA molecule that
transfers a DNA segment between host
cells.
Small
A cloning site (multi-clone site)
A replicator (origin of replication)
A selectable marker
Plasmid
An extrachromosomal genetic element
found in a variety of bacterial species.
Plasmid are double-stranded,closed DNA
molecules.
Plasmid
agtgaattCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGcgtaatcatggtcat
pUC19
2686 bp Ori
ApR
LacZ
EcoRⅠ
SacⅠ
KpnⅠ
SmaⅠ XbaⅠ
BamHⅠ SalⅠ SphⅠ
PstⅠ HindⅢ
Lambda Phage
A double-stranded DNA virus that is
engineered to serve as a receptor for
foreign DNA fragments in recombinant
DNA experiments.
Plasmid
Cos site
Lytic & Lysogenic
Replacement λ vector
Insertion λ vector
Cosmid
Vectors designed for cloned large
fragment of eukaryotic DNA,The term
signifies that the vector is a plasmid into
which phage lambda cos sites have been
inserted.
Cosmid
pJB8
5.4 kb
Sal Ⅰ
Others
P1 phage & PAC
BAC,bacterial artificial chromosome
YAC,yeast artificial chromosome
Centromere
Telomere
ARS,autonomous replication sequence
Back
Vector
Vector Targeting Fragment Length
Plasmid 0~10 kb
Replacement λ vector 0~10 kb
Insertion λ vector 9~23 kb
Cosmid 33~44 kb
P1 phage 70~100 kb
PAC 130~150 kb
BAC 300 kb
YAC 0.2~2.0 Mb
Nick Translation
p A p C p G p T p T p A p C p G p G p A p C p T p G p T p T p A p C p C p G p A p T
p T p G p C p A p A p T p G p C p C p T p G p A p C p A p A p T p G p G p C p T p A
DNaseⅠ DNA PolymeraseⅠ
dATP
dCTP
dGTP
dTTP
Nick Translation
p A p C p G p T p T p A C p G p G p A p C p T p G p T p T p A p C p C p G p A p T
p T p G p C p A p A p T p G p C p C p T p G p A p C p A p A p T p G p G p C p T p A
DNaseⅠ
dATP
dCTP
dGTP
dTTP
Nick Translation
p A p C p G p T p T p A C p G p G p A p C p T p G p T p T p A p C p C p G p A p T
p T p G p C p A p A p T p G p C p C p T p G p A p C p A p A p T p G p G p C p T p A
OH PDNA PolymeraseⅠ
dATP
dCTP
dGTP
dTTP
Nick Translation
p A p C p G p T p T p A p G p G p A p C p T p G p T p T p A p C p C p G p A p T
p T p G p C p A p A p T p G p C p C p T p G p A p C p A p A p T p G p G p C p T p A
dCMP
dATP
dCTP
dGTP
dTTP DNA
PolymeraseⅠ
p A p C p G p T p T p A p G p G p A p C p T p G p T p T p A p C p C p G p A p T
p T p G p C p A p A p T p G p C p C p T p G p A p C p A p A p T p G p G p C p T p A
pC
dATP
dCTP
dGTP
dTTP DNA
PolymeraseⅠ
Nick Translation
Back
Nick Translation
p A p C p G p T p T p A p C p G p G p A p C p T p G p T p T p A p C p C p G p A p T
p T p G p C p A p A p T p G p C p C p T p G p A p C p A p A p T p G p G p C p T p A
dATP
dCTP
dGTP
dTTP
DNA PolymeraseⅠ
Random-Primed Labeling
5’ 3’
5’3’
dATP dCTP
dGTP dTTP
Klenow
Klenow Klenow
Back
Random-Primed Labeling
5’ 3’
5’3’
