Chapter 4

Molecular Cloning Methods

Gene Cloning
The Role of Restriction
Endonuclease
Vectors
Plasmids :
carriers that allow replication
of recombinant DNAs
r: resistant
s: snsitive
Action of DNA ligase
lacZ: b-galactosidase
X-gal: synthetic substrate for b-galactosidase
MCS: multiple cloning sites

Blue/white color selection
Enhance of the cloning efficiency:

dephosphorylation of the 5-phophate in the vector by alkaline phosphatase
to prevent self-ligation
Phages as Vectors
replacement vectors

*up to 20 kb of DNA can be cloned.
*construction of genomic libraries
*Charon 4: (12 kb< inserts <20 kb)
Cosmids

* cohesive ends of phage DNA
* plasmid origin of replication
* up to 40-50 kb of DNA can be cloned
M13 phage vectors

* derived from filamentous phage M13
* contains lacZgene and MCS
* DNA can be cloned in a single-strand form
(introduction of site-specific mutation)
Phagemids

* contains ori of filamentous phage f1, lacZgene and MCS
* DNA can be cloned in a single-strand form via the help with f1 helper phage
* contains T7 and T3 phage promoter for phage RNA polymerase
(in vitro transcription to produce RNA transcripts)
Identifying a specific clone with a specific probe
1. Poly(oligo)nucleotide probes
2. Antibodies

To probe the gene what we want:

a. Homologous gene from another organism
*Low/high stringency for hybridization of oligonucleotide probes to targets
*High stringency: high temperature, high organic concentration,
low salt concentration

b. Degenerate primers from known amino acid sequence of target protein

U G U
UGG AUG UUC AAA AAC GA
Trp Met Phe Lys Asn Glu
The Polymerase Chain Reaction (PCR)
*Kary Mullis in the 1980s
*Taq polymerase from Thermus aquaticus
*Thermal cycler 95
o
C 40
o
C 72
o
C
20x
cDNA Cloning
(complementary or copy DNA)
Nick translation using
E. Coli DNA polymerase I
with 5 3 exonuclease
activity
Nick translation
Using RT-PCR in
cDNA Cloning
BamH1
HindIII
5-RACE of a cDNA
Rapid Amplification of cDNA Ends
(completing of the 5end of the mRNA)
Methods of Expressing
Cloned Genes
pUC and pBS vectorslac promoter
(in frame)
ptrpL 1 vectortrp operator/promoter
(strong promoter)
SD: Shine-Dargarno
ribosome binding site
Expression of
Hisx6-tagged fusion protein
using pTrcHis vector
EK: enterokinase (a protease) cutting site
Expression of fusion protein
using gt11 vector in E. Coli
Eukaryotic Expression
Systems
Expression in E.Coli:
Inclusion bodies
No posttranslational modification


Expression in yeast


Expression in caterpillar using
Baculovirus-derived vectors
(10% in dry mass, polyhedrin)