Chap. 4. Molecular cloning methods

- Gene Cloning

What is a CLONE ?

genotype

Monozygotic twin

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

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

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.

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

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

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 ?

Phagemid

Helper phage

cDNA libraryVs.

Genomic DNA library

DNA :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 ?

Kary Mullis

Denaturation (94 oC)Annealing (37-72 oC)Polymerization (72 oC)

Taq polymerase Thermus aquaticusThermal cycler = PCR machine

templateRT-PCR

Mbu-1 is specifically expressed in the mouse brain

A

RT-PCR

hear

tki

dney

lung

brai

nm

uscl

esp

leen

panc

reas

liver

test

is

mar

ker

Real-time RT-PCR

Re

lati

ve

le

ve

l

1.0

0.8

0.6

0.4

0.2

0

B

hear

tki

dney

lung

brai

nm

uscl

esp

leen

panc

reas

liver

test

is

A

1 5 10 15 20 25 30

Cycle Number

Del

ta R

n

Expression vectors

pUC19

Fusion protein

-Toxic - Inclusion body insoluble aggregates Inducible vector

pTrcHis

Affinity chromatography

cf. heme