Restriction Enzyme Analysis and Gel Electrophoresis of DNA
Learn how to cut DNA into fragments with restriction enzymes.
Load and separate DNA fragments by electrophoresis.
Determine the size of DNA molecules by use of a Standard Curve.
In this lab, we will analyze the DNA of small virus called a
Bacteriophages are bacterial viruses that can infect and kill some species of bacteria.
They have a simple, linear chromosome that is unique to that particular virus, and like
other viruses, can only attack and infect certain bacterial cells.
During infection of a bacterial cell, the virus inserts its genome into a bacterial cell in an
attempt to ‘hijack’ the bacterial cell’s protein synthesis (transcription and translation)
Bacteria can respond to the threat of bacteriophage infection by producing
that chop phage DNA up into small pieces, “restricting” viral infection.
Scientists first discovered these enzymes, called “
” in the late 1960s
and early 1970s.
(The bacteria’s own DNA is protected from digestion by
the A and C nucleotides in the DNA).
An interesting feature of many restriction enzymes
is their ability to recognize a sequence
of bases on DNA called a
defined as a stretch of DNA that ‘reads’
the same forwards (on one strand) as
they do ‘backwards’ on the
the figure at right shows the restriction
site from one of the first discovered
restriction enzymes, EcoRI (“
Restriction enzyme I”), which
recognizes the palindromic sequence
Many restriction enzymes
within the palindrome at a specific site called a "
", and the asymmetric cut results in small, single stranded regions called “
(TTAA in this case).
These sticky ends have been used by scientists to join or “splice”
together two pieces of DNA that have complementary sticky ends, for instance, to join the
human insulin gene
cut with EcoR1 to a bacterial plasmid cut with EcoR1, resulting in a
, made by Eli Lilly.
In this lab exercise, DNA from the
(48,502 base pairs in length)
will be cut with 3 different restriction enzymes: Pst1, EcoRI, and HindIII, and the
resulting fragments will separated by
. A fourth sample will be the
negative control in that is will be incubated without any restriction enzyme. Each of the
3 enzymes recognizes a different palindromic sequence of bases on the DNA, and cuts at
specific "restriction sites."
[HindIII recognizes the palindrome AAGCTT and PstI
recognizes the palindrome CTGCAG].