Chapter 4
Molecular Cloning Methods
Gene Cloning
The Role of Restriction Endonuclease
Vectors
Plasmids : carriers that allow replication of recombinant DNAs
r: resistants: snsitive
Action of DNA ligase
lacZ’: -galactosidaseX-gal: synthetic substrate for -galactosidaseMCS: 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 lacZ’gene and MCS
* DNA can be cloned in a single-strand form (introduction of site-specific mutation)
Phagemids
* contains ori of filamentous phage f1, lacZ’gene 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 probes2. 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 GATrp Met Phe Lys Asn Glu
The Polymerase Chain Reaction (PCR)
*Kary Mullis in the 1980s*Taq polymerase from Thermus aquaticus*Thermal cycler 95oC 40oC 72oC
20x
cDNA Cloning
(complementary or copy DNA)
Nick translation usingE. Coli DNA polymerase Iwith 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 5’end of the mRNA)
Methods of ExpressingCloned Genes
pUC and pBS vectors—lac promoter
(in frame)
ptrpL 1 vector—trp operator/promoter (strong promoter)
SD: Shine-Dargarno ribosome binding site
Expression of Hisx6-tagged fusion proteinusing pTrcHis vector
EK: enterokinase (a protease) cutting site
Expression of fusion proteinusing gt11 vector in E. Coli
Eukaryotic Expression Systems
Expression in E.Coli:Inclusion bodiesNo posttranslational modification
Expression in yeast
Expression in caterpillar using Baculovirus-derived vectors (10% in dry mass, polyhedrin)