chap. 4. molecular cloning methods
DESCRIPTION
Restriction endonuclease Haemophilus influemzae Rd; Hamilton Smith GTPyPuAC CAPuPyTG HindII plaque EcoRI Palindrome (rotational symmetry) 5’-GAATTC-3’ 3’-CTTAAG-5’ AATTC G Bacterial lawn G CTTAA Sticky end ; protruding, cohesive, overhang Average length ; 46 = 4,096 bpTRANSCRIPT
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Chap. 4. Molecular cloning methods
- Gene Cloning
What is a CLONE ?
genotype
Monozygotic twin
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GTPyPuACCAPuPyTG
Haemophilus influemzae Rd; Hamilton Smith
HindII
EcoRI
5’-GAATTC-3’3’-CTTAAG-5’
GCTTAA
AATTCG
Sticky end ; protruding, cohesive, overhang
Average length ; 46 = 4,096 bp
Palindrome(rotational symmetry)
Restriction endonuclease
Bacterial lawn
plaque
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1. Restirction endonuclease 2. ligase
- Use ATP for formation of phosphodiester bond
Sticky end ; 5’-protruding, EcoRI : 3’-protruding, PstIBlunt end; SmaIHeteroschizomer ; SmaI, XmaI cf) isoschizomer
5’-P3’-OH
5’-P
3’-OH
EcoRI
PstI
SmaI
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Restriction-modification system
Bacterial lawn
plaque
-CH3Methylases methylate the recognition sites for
restriction endonuclease to protect the host DNA.
The methylation is conserved during replication
process.
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Vectorplasmid pBR322
The first letter or plasmid should be noted by lower case “p”.
vector insert
1.Replication origin2.Selection marker ; antibiotic resistance gene3.Multicloning sites (MCS)
pUC19
Ampicillin, X-gal, IPTGβ-galactosidase
Self-ligationClone cloning
- Dephosphorylation ; Alkaline phosphatase - directional cloning ; compatible
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LyticLysogenic
Vs. colony
Average insert size ; ~ 15 kb
Bacterial lawnplaque
Disadvantages of plasmid- Size - transformation efficiency
Phage vector
Plaque hybridization
colony hybridizationLibrary, screening
P P PP
Homologous gene
혼성화탐침자가방사법
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Probe ;1. Polynucletide probe ; homologous gene stringency; low or high
- temp.- denaturing agent ; - ionic strength
2. Oligonucleotide probe ; 17-mer, 21-mer
- degenerated primer U G U
UGG AUG UUC AAA AAC GA W M F K N Q
Cosmid, YAC, BAC; Average length ?
PhagemidHelper phage
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cDNA libraryVs.
Genomic DNA libraryDNA :RNA hybrid
5’3’ polymerization 5’3’ exonuclease 3’ 5’ exo.
Nick translation
Could we directly clone a specific genomic sequence
or cDNA by PCR without screening ?
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Kary Mullis
Denaturation (94 oC)Annealing (37-72 oC)Polymerization (72 oC)
Taq polymerase Thermus aquaticusThermal cycler = PCR machine
templateRT-PCR
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Mbu-1 is specifically expressed in the mouse brain
A
RT-PCR
hear
tkid
ney
lung
brain
mus
clesp
leen
panc
reas
liver
testi
s
mar
ker
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Real-time RT-PCR
Rel
ativ
e le
vel
1.0
0.8
0.6
0.4
0.2 0
B
hear
tkid
ney
lung
brain
mus
clesp
leen
panc
reas liver
testi
s
A
1 5 10 15 20 25 30
Cycle Number
Del
ta R
n
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Expression vectors
pUC19
Fusion protein
-Toxic - Inclusion body insoluble aggregates Inducible vector
pTrcHis
Affinity chromatography
cf. heme