Download Why Clone DNA?

Cloning and Vector
Chapter 3
Instructor : Prof. Myoung-Dong Kim
T: 6458, [email protected]
Room 411, Ag. Bld #3
Gene Cloning
Cloning - a definition
 From the Greek - klon, a twig
 An aggregate of the asexually produced progeny
of an individual;a group of replicas of all or part
of a macromolecule (such as DNA or an
antibody)
 An individual grown from a single somatic cell of
its parent & genetically identical to it
 Clone: a collection of molecules or cells, all
identical to an original molecule or cell
DNA CLONING
A method for identifying and purifying a
particular DNA fragment (clone) of interest
from a complex mixture of DNA fragments,
and then producing large numbers of the
fragment (clone) of interest.
Gene cloning
 When DNA is
extracted from an
organism, all its
genes are obtained
 In gene (DNA)
cloning a particular
gene is copied
(cloned)
Why Clone DNA?
 A particular gene can be isolated and its
nucleotide sequence determined
 Control sequences of DNA can be identified &
analyzed
 Protein/enzyme/RNA function can be
investigated
 Mutations can be identified, e.g. gene defects
related to specific diseases
 Organisms can be ‘engineered’ for specific
purposes, e.g. insulin production, insect
resistance, etc.
Sources of DNA for Cloning
 1) Chromosomal DNA
 2) RNA converted to cDNA
 3) PCR-amplified DNA
PCR-amplified DNA
Cloning Tools





Restriction endonucleases
Ligase
Vectors
Host
Methods for introducing DNA into a
host cell
Cutting DNA
 Restriction endonucleases
(restriction enzymes)
• sticky ends
• blunt ends
 Nomenclature
•
•
•
•
•
EcoRI
E = genus (Escherichia)
co = species (coli)
R = strain
I = # of enzyme
Blunt & Sticky ends
Pasting DNA
 Complementary
ends (sticky
ends) H-bond
 Ligase forms
phosphodiester
bond to seal
strands together.
Vectors
Cloning vectors
Allowing the exogenous DNA to be
inserted, stored, and manipulated
mainly at DNA level.
1 Plasmid vectors
2 Bacteriophage vectors
3 Cosmids
4 BACs & YACs
Plasmid vectors
Plasmid vectors are double-stranded, circular, selfreplicating, extra-chromosomal DNA molecules.
 Advantages:
• Small, easy to handle
• Straightforward selection strategies
• Useful for cloning small DNA fragments
(< 10kbp)
 Disadvantages:
• Less useful for cloning large DNA fragments
(> 10kbp)
Plasmid vectors
 Plasmids are circular DNA
molecules present in the
cytoplasm of the bacteria
 Capable of autonomous
replication
 Can transfer genes from
one cell to other
 Act as vectors in genetic
engineering.
 Can also present in
Yeasts
Plasmid vectors
 may encode genetic information for properties
1 Resitance to Antibiotics
2 Bacteriocins production
3 Enterotoxin production
4 Enhanced pathogen city
5 Reduced Sensitivity to mutagens
6 Degrade complex organic molecules
T.V.Rao MD
Plasmid vector for cloning
1. Contains an origin of replication, allowing for
replication independent of host’s genome.
2. Contains Selective markers: Selection of cells
containing a plasmid
twin antibiotic resistance
blue-white screening
3. Contains a multiple cloning site (MCS)
4. Easy to be isolated from the host cell.
Plasmid vectors
Bacteriophage vectors
 Advantages:
• Useful for cloning large DNA fragments
(10 - 23 kbp)
• Inherent size selection for large inserts
 Disadvantages:
• Less easy to handle
l vectors
 Left arm:
• head & tail proteins
 Right arm:
• DNA synthesis
• regulation
• host lysis
 Deleted central region:
• integration &
excision
• regulation
Bacteriophage
Cosmid vectors
Combine the properties of plasmid vectors
with the useful properties of the l cos site
 Advantages:
• Useful for cloning very large DNA
fragments
(32 - 47 kbp)
• Inherent size selection for large inserts
• Handle like plasmids
 Disadvantages:
• Not easy to handle very large plasmids
• (~ 50 kbp)
l ZAP
BACs and YACs
BACs : Bacterial Artificial Chromosomes
YACs : Yeast Artificial Chromosomes
 Advantages:
• Useful for cloning extremely large DNA fragments
(100 - 2,000 kbp)
• This is very important for genome sequencing
projects
 Disadvantages:
• Not easy to handle extremely large DNA
molecules
BAC vector
 oriS and oriE
mediate replication
 parA and parB
maintain single
copy number
 ChloramphenicolR
marker
YAC vector
large
inserts
ARS
URA3
telomere
centromere
replication
origin
HIS3
markers
telomere
 Capable of carrying inserts of 200 - 2000 kbp in yeast
What determines the choice vector?
 insert size

vector size

restriction sites

copy number

cloning efficiency

ability to screen for inserts

what down-stream experiments do you plan?
Expression vector
Expression vector pSE420
• Polylinker: insert desired DNA
• Amp resistance
• trc promoter
• lacO (operator)
• Shine-Dalgarno (S/D) site
(ribosome binding)
• T1, T2 transcription
terminators
• lacI (lac repressor)
growth
inducer added
cloned gene
expressed;
product produced
Btech6
How to clone DNA
• insertion of foreign DNA
at BamHI site
• tet resistance gene
inactivated
• transformants carrying
foreign DNA are amp
resistant but tetracycline
sensitive
transformation: transfer
of genetic information
via free DNA
How to clone DNA





Isolation of cloning vector
(bacterial plasmid) & genesource DNA (gene of interest)
Insertion of gene-source DNA
into the cloning vector using
the same restriction enzyme;
bind the fragmented DNA with
DNA ligase
Introduction of cloning vector
into cells (transformation by
bacterial cells)
Cloning of cells (and foreign
genes)
Identification of cell clones
carrying the gene of interest
Screening of the clone
 The medium in this petri dish
contains the antibiotic
Kanamycin
 The bacteria on the right
contain Kanr, a plasmid that is
resistant to Kanamycin, while
the one on the left has no
resistance
 Note the difference in growth
Blue/White Color Screening
lacZ
lacZ
functional enzyme
X-gal
product
insert
nonfunctional enzyme
X-gal
product
Selecting Colonies with Recombinant
Plasmids
Colony hybridization
 DNA probe available?
• part of same gene
• orthologue from another
species
• synthetic oligonucleotide
Bacteriophage lambda as a cloning vector
transduction: transfer of host genes from one cell to another by a virus
(Fig. 10.44, p. 311, Madigan et al.)
Other methods for introducing DNA
Electroporation: the use of an electric pulse to enable cells to take up DNA
• Millisecond-length pulses open small pores in cell membranes
• DNA can move into/out of the cells via pores
cell
plasmid donor
microprojectile “gun”
plasmid
transformant
desired transformant
Transgenic plants may be produced with binary vector system in
Agrobacterium tumefaciens
(a) generalized plant cloning vector
• ends of T-DNA (red)
• ori (E. coli), ori (A. tumefaciens)
• resistance markers (kan, spec)
(b) can clone in E. coli; transfer to
A. tumefaciens by conjugation
(c) D-Ti = engineered Ti (to remove
pathogenesis genes)
(d) D-Ti will mobilize T-DNA of
vector → plant cells grown in
tissue culture
(e) whole plants can be regenerated
from recombinant cell