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Lecture 5:
Restriction Endonucleases
History
Definition
Enzyme that binds to a DNA molecule at a
specific sequence and makes a doublestranded cut at or near that sequence
Sequence specific, the position of cutting
within a DNA molecule can be predicted
Types of RE
Type I: complex, multisubunit, combination restrictionand-modification enzymes that cut DNA at random far
from their recognition sequences
Type II: cut DNA at defined positions close to or within
their recognition sequences
Type III: cleave outside of their recognition sequences
and require two such sequences in opposite orientations
within the same DNA molecule to accomplish cleavage; they
rarely give complete digests
Type IV: recognize modified, typically methylated DNA
Nomenclature
Based upon the name of the organism from which they
were isolated
PstI:
The letter I, II and III indicate the first, second and third RE
isolated from H. aegyptius respectively
Mode of Action
1.
Hydrogen
bonds
Nick
2.
The bonds being broken by the enzyme are covalent. The hydrogen bonds
responsible for base pairing are not broken by the restriction enzyme
3.
Isoschizomers
5 mM Magnesium chloride
10 mM Magnesium acetate
100 mM NaCl
66 mM Potassium acetate
1 mM 2-mercaptoethanol
0.5 mM Dithiothreitol
Reaction conditions: 37 C
Reaction conditions: 25 C
Star Activity
It has been demonstrated that under extreme
non-standard conditions, restriction
endonucleases are capable of cleaving sequences
which are similar but not identical to their
defined recognition sequence
The altered or relaxed specificity has been
termed ""star"" activity
Class Activity
Class Activity
Class Activity
End of Lecture 5
Thank You
Lecture 6:
Gene Cloning and Analysis