Documente Academic
Documente Profesional
Documente Cultură
Sawitree Chiampanichayakul
Dept. of Medical Technology
Fac. of Associated Medical Sciences
Chiang Mai University
DNA Cloning – the act of making many identical copies
of a particular piece of DNA (often a gene).
DNA Cloning
Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
Overall of DNA Cloning
Isolation of
DNA
Selection of cloning
vector act as a vehicle
Vector DNA
Plasmid DNA
Phage DNA
Genomic DNA Libraries
Genomic library is all DNA, introns, exons and non-coding
Foreign genome
cut up with
restriction
enzyme
or
Bacterial Recombinant
plasmids Recombinant
clones phage DNA Phage
clones
Linear DNA
pH 12-12.5
Supercoiled Single-stranded
plasmids linear DNA
pH 7
Tangled mass of
linear DNA
Ethidium bromide-Caesium chloride density gradient centrifugation
CsCl CsCl+EtBr
Protein Protein
1.6
1.65
1.7 DNA Linear DNA
1.75
Supercolied DNA
1.8
RNA RNA
How are the pieces of DNA put back together?
Restriction enzyme
Sticky end
Restriction enzyme Type II
DNA 5′ GAATTC 3′
3′ CTTAAG
5′
1 Restriction enzyme cuts
the sugar-phosphate
backbones at each arrow
G AATTC
CTTAA G
Sticky end
2 DNA fragment from
another source is added. AATTC
G
Base pairing of sticky G
CTTAA
ends produces various
combinations. Fragment from different
DNA molecule cut by the
same restriction enzyme
G AATT C G AATTC
C TTAA G CTTAA G
Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
Cloning Vectors
Polylinker:
restriction
sites
lacZ+
gene
Origin
Ampicillin sequence
resistance
gene
Fig. 7.5
*Cut with same
restriction enzyme
*DNA ligase
Bacteriophage cloning vector
Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
Creating Recombinant DNA
A plasmid vector is digested with EcoRI at a single site to
produce two sticky ends.
Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
Inserting vectors into host cells
Isolation of DNA
Selection of cloning vector act as a vehicle
Ligating the DNA into a vector to make recombinant DNA
Introduction of recombinant DNA into host cells
Selection of host cells containing the recombinant DNA
Some possible ligation reaction products:
Phenotyping
Colony hybridization
To selectively kill cells, and select for successfully transformed
bacterial cells, you must screen the original bacterial colonies
from the original master plate, and make exact copies by
replica plating :
The clones are blot transferred to a membrane sheet, and the DNA
present denatured and fixed onto the surface.
Recombinate
bacterium
3
3 Host cell grown in culture,
to form a clone of cells
containing the “cloned”
gene of interest
Gene of
interest Protein expressed
by gene of interest
Copies of gene Protein harvested
Basic Basic
research 4 Basic research and research
on gene various applications on protein
Gene for pest Gene used to alter Protein dissolves Human growth
resistance inserted bacteria for cleaning blood clots in heart hormone treats
into plants up toxic waste attack therapy stunted growth