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Cloning and Vector: Instructor: Prof. Myoung-Dong Kim T: 6458, Mdkim@kangwon - Ac.kr Room 411, Ag. BLD #3
Cloning and Vector: Instructor: Prof. Myoung-Dong Kim T: 6458, Mdkim@kangwon - Ac.kr Room 411, Ag. BLD #3
Cloning and Vector: Instructor: Prof. Myoung-Dong Kim T: 6458, Mdkim@kangwon - Ac.kr Room 411, Ag. BLD #3
Chapter 3
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
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, self-
replicating, 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
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
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
insert size
vector size
restriction sites
copy number
cloning efficiency
• trc promoter
• lacO (operator)
• Shine-Dalgarno (S/D) site
(ribosome binding)
• T1, T2 transcription
terminators
• lacI (lac repressor)
cloned gene
growth inducer added expressed;
product produced
Btech6
transformation: transfer
of genetic information
via free DNA
How to clone DNA
microprojectile “gun”
Transgenic plants may be produced with binary vector system in
Agrobacterium tumefaciens