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Restriction Enzymes

Restriction enzymes recognize specific short DNA sequences called restriction sites and cleave DNA molecules at these sites. The enzyme BamHI recognizes the palindrome sequence 5'-GGATCC-3' and cuts within this sequence, leaving "sticky ends". Restriction enzymes can cut DNA asymmetrically, leaving overhangs, or symmetrically at the same position on each strand. They cut DNA from any source as long as the restriction site is present. This cleavage produces restriction fragments that can be analyzed by gel electrophoresis.

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204 views9 pages

Restriction Enzymes

Restriction enzymes recognize specific short DNA sequences called restriction sites and cleave DNA molecules at these sites. The enzyme BamHI recognizes the palindrome sequence 5'-GGATCC-3' and cuts within this sequence, leaving "sticky ends". Restriction enzymes can cut DNA asymmetrically, leaving overhangs, or symmetrically at the same position on each strand. They cut DNA from any source as long as the restriction site is present. This cleavage produces restriction fragments that can be analyzed by gel electrophoresis.

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Restriction Enzymes and SiteSpecific DNA Cleavage

a class of enzymes known as restriction endonucleases or


restriction enzymes are able to cleave DNA molecules at the
positions at which particular, short sequences of bases are
present.
For example, the enzyme BamHI recognizes the double-stranded
sequence
5'-GGATCC-3'
3'-CCTAGG-5'

Mechanism of DNA cleavage by the restriction enzyme BamHI. The enzyme


makes a single cut in the backbone of each DNA strand wherever the duplex
contains a BamHI restriction site

The nucleotide sequence recognized for cleavage by a restriction


enzyme is called the restriction site of the enzyme

The restriction enzymes cleave their restriction site asymmetrically


(at different sites in the two DNA strands), but some restriction
enzymes cleave symmetrically (at the same site in both strands).
The former leave sticky ends because each end of the cleaved site
has a small, single-stranded overhang that is complementary in base
sequence to the other end.

In virtually all cases, the restriction site of a restriction enzyme


reads the same on both strands, provided that the opposite polarity
of the strands is taken into account; for example, each strand in the
restriction site of BamHI reads 5'-GGATCC-3.
A DNA sequence with this type of symmetry is called a
palindrome. (In ordinary English, a palindrome is a word or
phrase that reads the same forwards and backwards, such as
''madam.")

Table -Some restriction endonucleases, their sources, and their cleavage sites

Note: The vertical dashed line indicates the axis of symmetry in each sequence. Red
arrows indicate the sites of cutting. The enzyme TaqI yields cohesive ends consisting of
two nucleotides, whereas the cohesive ends produced by the other enzymes contain four
nucleotides. Pu and Py refer to any purine and pyrimidine, respectively.

Restriction enzymes have the following important


characteristics:
Most restriction enzymes recognize a single restriction site.
The restriction site is recognized without regard to the source of
the DNA.
Because most restriction enzymes recognize a unique restriction
site sequence, the number of cuts in the DNA
from a particular organism is determined by the number of
restriction sites present.

The DNA fragment produced by a pair of adjacent cuts in a


DNA molecule is called a restriction fragment. A large
DNA molecule will typically be cut into many restriction
fragments of different sizes.
For example, an E. coli DNA molecule, which contains 4.7
106 base pairs, is cut into several hundred to several
thousand fragments, and mammalian nuclear DNA is cut
into more than a million fragments

A map showing the unique sites of cutting of the DNA of a


particular organism by a single enzyme is called a restriction
map.
The family of fragments produced by a single enzyme can be
detected easily by gel electrophoresis, and particular DNA
fragments can be isolated by cutting out the small region of the
gel that contains the fragment and removing the DNA from the
gel.

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