Restriction enzymes: a glimpse at their use in genetic engineering

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11 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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Restriction enzymes:

a glimpse at their use in genetic engineering


"Restriction endonuclease" is the name of a type of enzyme that cleaves both strands of DNA
molecules at specific sites. They are sometimes called "restriction enzymes." There are many
e
nzymes in this group, each of which recognizes a different, specific short sequence of base pairs
in the DNA molecule. Wherever that sequence of base pairs occurs within the DNA molecule,
the enzyme cleaves the sugar
-
phosphate backbone of each strand.


The enzymes probably originated in primitive prokaryotes as a means of protection against
viruses. The
enzymes would destroy viral DNA when it entered the bacterial cell.


In the typical situation the enzyme's recognition site on the DNA molecule is a
pal
indrome
,
which is a sequence that reads the same backward as forward. The famous quotation "Able was
I ere I saw Elba" is an example. Many single words are palindromes: kayak, civic, toot, pop, for
example. Figure A in the drawing below shows a sequence

of DNA base pairs that is a
palindrome. When read from left to right, the pairs are AT, TA, GC, CG, GC, CG, AT, TA. The
sequence is the same when read from right to left.


What made the
se enzymes
interesting was the fact that they cleave the DNA double
helix
asymmetrically,
i.e.
not straight across. Figure B below shows how one of these enzymes, after
recognizing the 8
-
base pair palindrome, would cleave at the locations marked by the arrows.
After the pentose
-
phosphate backbones have been broken at tho
se two spots, the hydrogen bonds
between the bases of those 8 pairs are readily broken (recall that H
-

bonds are weak). The result:
the double helix fractures at that site, with each strand having a short sequence of unpaired bases,
called "sticky ends,"
as shown in Figure C below.


Now, suppose we extract DNA from cells of two different species and treat both DNA samples
with the same restriction enzyme. Regardless of what species the DNA is from, the enzyme will
cleave DNA molecules at the same site,
i.e. that palindrome. Figure D below shows, for
example, that the palindrome occurs at 2 places in one DNA molecule and one place in the other
DNA molecule. After treatment of the DNA with the enzyme, let's mix the two samples of DNA
fragments and then u
se DNA ligase to reattach the pentose
-
phosphate backbones of the DNA
molecules.


Because the sticky ends are the same in the DNA fragments from both species, there will be
some matching up of fragments from different species' DNA. The result will be some
newly
reconstituted DNA molecules that contain segments from both species, as shown in Figure D, far
right. Since DNA fragments bear genes, the new "hybrid" DNA molecule contains one or more
genes that it did not have originally.


You will have an opportu
nity to learn some of the details of how this procedure is done in future
coursework. Here you see just a hint of how DNA might be "engineered," i.e. altered at will.
Such insertion of genes into a DNA molecule in a cell would give that cell the ability t
o make
proteins that it could not make before.