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

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

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

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

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

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

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

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

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

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

A B Real-time RT-PCR Delta Rn Cycle Number 1 5 10 15 20 25 30 heart 1 5 10 15 20 25 30 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

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