Chapter 4 Molecular Cloning Methods. Gene Cloning The Role of Restriction Endonuclease.

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)