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BIOTECHNOLOGY
CONTENT
• Plasmids
• Cosmids
• Phages
• Yeast Artificial Chromosomes
(YACs)
• Bacterial Artificial Chromosomes
(BACs)
Plasmids
• Double stranded, circular DNA which exist in
bacteria, yeast and organelles
• May exist as single copy per cell or multi-
copy
per cell (10-20 genomes/cell), or even under
relaxed replication control where up to 1000
copies/cell can be maintained
• Size of rDNA insertions limited to ~10kb
PLASMIDS
• Bacterial cells may
contain extra-
chromosomal DNA
called plasmids.
• Plasmids are usually
represented by small,
circular DNA.
• Some plasmids are
present in multiple
copies in the cell
PLASMID VECTORS
Plasmid vectors are ≈1.2–3kb
and contain:
• replication origin (ORI)
• a gene that permits selection,
• Here the selective gene is
ampr; it encodes the enzyme
b-lactamase, which inactivates
ampicillin.
• Exogenous DNA can be
inserted into the bracketed
region .
SELECTIVE MARKER
• Selective marker is required for maintenance of
plasmid in the cell.
• Because of the presence of the selective marker
the plasmid becomes useful for the cell.
• Under the selective conditions, only cells that
contain plasmids with selectable marker can
survive
• Genes that confer resistance to various
antibiotics are used.
• Genes that make cells resistant to ampicillin,
neomycin, or chloramphenicol are used
ORIGIN OF REPLICATION
• Origin of replication
is a DNA segment
recognized by the
cellular DNA-
replication enzymes.
• Without replication
origin, DNA cannot
be replicated in the
cell.
MULTIPLE CLONING SITE
• A DNA segment with several
unique sites for restriction
endo- nucleases located next
to each other
• Restriction sites of the
polylinker are not present
anywhere else in the plasmid.
• Cutting plasmids with one of
the restriction enzymes that
recognize a site in the
polylinker does not disrupt
any of the essential features of
the vector
MULTIPLE CLONING SITE
E Escherichia genus
co coli species
R RY13 strain
order of identification
I First identified
in the bacterium
B. Availability
• Type I
– Cuts the DNA on both strands but at a non-
specific location at varying distances from
the particular sequence that is recognized by
the restriction enzyme
– Therefore random/imprecise cuts
– Not very useful for rDNA applications
• Type II
– Cuts both strands of DNA within the
particular sequence recognized by the
restriction enzyme
– Used widely for molecular biology
procedures
– DNA sequence = symmetrical
• Reads the same in the 5’ 3’ direction on
both strands = Palindromic Sequence
• Some enzymes generate “blunt ends” (cut in
middle)
• Others generate “sticky ends” (staggered
cuts)
– H-bonding possible with complementary tails
– DNA ligase covalently links the two fragments together
by forming phosphodiester bonds of the phosphate-
sugar backbones
Hae III
HaeIII is a restriction enzyme that searches the DNA
molecule until it finds this sequence of four nitrogen
bases.
5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
Once the recognition site is found Hae III will cleave
the DNA at that site
5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
These cuts produce
“blunt ends”
5’ TGACGGGTTCGAGG CCAG 3’
3’ ACTGCCCAAGGTCC GGTC 5’
“blunt ends” and “sticky ends”
Hae III produced a “blunt end”?
EcoRI makes a staggered cut and produces a
“sticky end”
5’ GAATTC 3’
3’ CTTAAG 5’
5’ GAATTC 3’
3’ CTTAAG 5’
5’ G AATTC 3’
3’ CTTAA G 5’
More examples of restriction sites of restriction
enzymes with their cut sites
Alu I: 5’ AGCT 3’
3’ TCGA 5’
• Some recognise very short sequences
consisting of only 4 base pairs. These
tend to cut DNA more frequently
(generating smaller fragments)
• Some require longer recognition
sequences (up to 8 bp). The longer
the recognition sequence the less
frequently these sites are likely to
occur
DNA LIBRARIES
Screening DNA Libraries
• Probe hybridization screens
– Using antibody probes to detect antigenic
fusion proteins -
• Protein-specific antibodies can be used as
"probes" to immunologically detect bacterial
fusion proteins encoding the
protein antigen.
Vectors such as λgt11 and lambda Zap
phage can be used.