1. Chap. 4. Molecular cloning methods
- Gene Cloning
What is a CLONE ?
genotype
Monozygotic twin

2. 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 bp

3. 1. Restirction endonuclease
2. ligase
Use ATP for formation of phosphodiester bond
5’-P
3’-OH
EcoRI
PstI
SmaI
Sticky end ; 5’-protruding, EcoRI : 3’-protruding, PstI
Blunt end; SmaI
Heteroschizomer ; SmaI, XmaI cf) isoschizomer

4. Restriction-modification system
Bacterial lawn
plaque
Methylases methylate the recognition sites for restriction endonuclease to protect the host DNA. The methylation is conserved during replication process.
-CH3

5. Vector plasmid pBR322 Ampicillin, X-gal, IPTG β-galactosidase
pUC19
The first letter or plasmid should be noted by lower case “p”.
Dephosphorylation ;
Alkaline phosphatase
directional cloning ;
compatible
vector
insert
1.Replication origin
2.Selection marker ; antibiotic resistance gene
3.Multicloning sites (MCS)
Ampicillin, X-gal, IPTG
β-galactosidase
Self-ligation
Clone  cloning

6. Phage vector Plaque hybridization colony hybridization
Disadvantages of plasmid
Size
transformation efficiency
Plaque hybridization
colony hybridization
Library, screening
Lytic
Lysogenic
Average insert size ;
~ 15 kb P P P P
Homologous
gene
Bacterial lawn
plaque
Vs. colony
혼성화 탐침
자가방사법

7. Phagemid
Helper phage
Cosmid, YAC, BAC
; Average length ?
Probe ;
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

8. 5’3’ polymerization 5’3’ exonuclease 3’ 5’ exo.
cDNA library
Vs.
Genomic DNA library
DNA :
RNA hybrid
Nick translation
5’3’ polymerization ’3’ exonuclease 3’ 5’ exo.
Could we directly clone a specific genomic sequence
or cDNA by PCR without screening ?

9. Kary Mullis
Denaturation (94 oC)
Annealing (37-72 oC)
Polymerization (72 oC)
Taq polymerase
Thermus aquaticus
Thermal cycler = PCR machine
template
RT-PCR

10. Mbu-1 is specifically expressed in the mouse brain
RT-PCR
heart
kidney
lung
brain
muscle
spleen
pancreas
liver
testis
marker

11. A B Real-time RT-PCR Delta Rn Cycle Number 1 5 10 15 20 25 30 heart
Cycle Number
Delta Rn
Relative level
1.0
0.8
0.6
0.4
0.2 B
heart
kidney
lung
brain
muscle
spleen
pancreas
liver
testis

12. Affinity chromatography
Expression vectors
pUC19
-Toxic
- Inclusion body
insoluble aggregates
 Inducible vector
Fusion protein
pTrcHis
Affinity chromatography
cf. heme