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Is it:
some possibilities
The recognition site for one enzyme may contain the restriction
site for another. The BamHI recognition site contains the
recognition site for Sau3AI.
restriction fragment
DNA fragment produced by a pair of
adjacent cuts in a DNA molecule
important properties
have an “ori” - origin of DNA
replication
a dominant selectable marker – enables
cells that carry the plasmid to be
distinguished easily
unique restriction cleavage sites – sites
present just one in the vector for
insertion of the DNA fragments
.
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Vectors
common vectors
E coli plasmids
bacteriophages (virus that attacks
bacteria)
lambda ()
M13
cosmids
plasmid with cohesive ends of phage
artificial chromosomes .
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Plasmids
Plasmid- small, circular,
extrachromosomal
DNA which replicates
independently of host
chromosomal DNA.
Most (experimental)
derived from a single
clinical specimen in
1974
Low copy # vs. high copy
number
Incompatible plasmids
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Isolation of Plasmid DNA
• Lysis by boiling
• Alkaline lysis
• Detergents or organic solvents
• Ori
• antibiotic
resistance gene(s)
• restriction sites
Bacterial chromosome
(DNA)
Figure 12.1A
Phage
Fragment of
DNA from Sex pili
another
bacterial cell
(former phage
host)
Crossovers
Figure 12.1D
Male (donor)
cell
plasmid, a small circular
Bacterial
chromosome DNA molecule separate
F factor starts
from the bacterial
replication and
transfer chromosome
Plasmids
Plasmid completes
transfer and
circularizes
Recombinant
bacterium
• Restriction enzymes
recognition sequence
1
cut DNA at specific
DNA
points
Restriction enzyme
cuts the DNA into
fragments
together
another source
Two (or more)
fragments stick
together by
• The result is 4
base-pairing
recombinant DNA
DNA ligase
pastes the strand
5
Figure 12.4 Recombinant DNA molecule
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings
Genes can be cloned in recombinant plasmids:
A closer look
2 Cut both
DNAs with
Plasmid the same
restriction DNA
enzyme
Gene V
Sticky ends
Recombinant DNA
plasmid Gene V
technology allows
enzyme
Recombinant
plasmid Recombinant
the construction of OR
phage DNA
genomic libraries
– Genomic libraries are
sets of DNA fragments
containing all of an
Phage
Bacterial clone
clone
organism’s genes
Plasmid Phage
processed called
• pGEM-3Z, e.g.
– Ampr
– lacZ
– polycloning site in lacZ gene
– T7 promoter one side, SP6 other