Extension-- Plasmid Recombination

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12 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

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Extension
--

Plasmid Recombination


USING RESTRICTION ENZYMES

The DNA sequences used in the construction of recombinant DNA molecules can originate from
any

species. For example, plant DNA may be joined to bacterial DNA, or human DNA may be
joined with
fungal DNA. In addition, DNA sequences that do not occur anywhere in nature may be
created by the

chemical synthesis of DNA, and incorporated into recombinant molecules. Using
recombinant DNA technology and synthetic DNA, literally any DNA sequence may be
created and
introduced into any of a very wide range of living organisms.


Gene cloning and genetic engineering were made possible by the discovery of enzymes that cut
DNA molecules at a limited number of specific locations. These enzymes, called restrict
ion
enzymes, were discovered in bacteria in the late 1960s. In nature, these enzymes protect the bacteria
against intruding DNA from other organisms, such as viruses or other bacterial cells. They work by
cutting up the foreign DNA, a process called rest
riction. Most restriction enzymes are very specific,
recognizing short nucleotide sequences in DNA molecules and cutting at specific points within these
sequences. The bacterial cell protects its own DNA from restric
tion by adding methyl groups
(
--
CH3) to adenines or cytosines within the sequences recognized by restriction enzymes. Hundreds
of different restriction enzymes have been identified and isolated.


Most restriction sites are symmetrical. The same sequence of four
to eight nucleotides
is found on both strands but running in
opposite directions. Restriction enzymes cut the bonds of both
strands, often in a staggered way. Since the target sequence usually
occurs many times in a long DNA molecule, an enzyme will make
many cuts. Copies o
f a DNA molecule always yield the same set of
restriction fragments when exposed to that enzyme. In other words,
a restriction enzyme cuts a DNA molecule in a reproducible way.


Notice that the restriction fragments are double
-
stranded DNA
fragments wit
h at least one single
-
stranded end, called a sticky end.
These short extensions will form hydrogen
-
bonded base pairs with
complementary single
-
stranded stretches on other DNA molecules
cut with th
e same enzyme. The unions formed

in this way are only
temp
orary, because only a few hydrogen bonds hold the fragments
together. The DNA fusions can be made permanent, however, by
the enzyme DNA ligase, which seals the strands by forming
permanent bonds.


MAKING RECOMBINANT DNA VECTORS

With a restriction enzyme

and DNA ligas
e, we can make a

recombinant plasmid
. The original
plasmid
is called a cloning vector, defined as a DNA molecule that can carry foreign DNA into a cell
and replicate there. Bacterial plasmids are widely use
d

as cloning vectors. Recombinant

plasmids
produced by splicing restriction fragments from foreign DNA into plasmids isolated from bacteria
can be returned relatively easily to bacteria. Then, as a bacterium carrying a recombinant plasmid
reproduces, the plasmid
is
replicated within it.

The resulting cell clone, which appears as a colony,
contains multiple copies of the foreign DNA.

Reflection Questions


1. Why do bacteria have restriction
enzymes?



1a. Why don’t the restriction enzymes cut the
bacteria’s own DNA?




2. Thi
nk of a symmetrical pattern that a restriction enzyme
might use as a cut site. It must read the same going forward on
the top strand and backwards on the bottom strand.




3. Most restriction enzymes cut in a way that is different than
we learned in cl
ass. How do most restriction enzymes cut?



3a. Why is this cut pattern important?






4. Which enzyme is used to glue the DNA pieces together?



5. Why are both the plasmid and the gene of interest cut using
the same restriction enzyme?






6. What are some recombinations that could easily happen to
result in a plasmid being created that does NOT contain your
desired result? Think of at least 2.







7. When would PCR be used in the process of creating a
recombinant plasmid?




Go to the
se websites to learn more:

http://www.dnalc.org/view/16723
-
Problem
-
34
-
Genes
-
can
-
be
-
moved
-
between
-
species
-
.html

http://www.youtube.com/watch?v=8rXizmLjegI