Fundamentals of Molecular Biotechnology (BIOL4900)

Department of Biology
Memorial University of Newfoundland

Essential Properties of Restriction Enzymes

DNA restriction and modification systems are common mechanisms by which bacteria protect their DNA from contamination by invading or foreign DNA.
Many bacteria produce a restriction enyzme that cleaves foreign DNA at a specific sequence.
For the bacteria to survive the presence of these enzymes, an accompanying DNA modification system (such as adenine methylation and cytosine methylation)

Type II restriction/modification systems consist of two separate proteins:
    1) a restriction endonuclease and
    2) a separate methylase.
Note that Type I and III restriction/modification systems have both activities included within one protein.

Restriction enzymes bind to and cleave double-stranded DNA at specific sites.
Different restriction sites recognize different sequences.

Most type II restriction enzymes recognize symmetric sequences that are 4, 5 or 6 base pairs in length.
A small minority of restriction enzymes recognize larger sequences.
Some recognize somewhat non-symmetric sequences.
The site of cleavage within the recognition sequence can produce
    1) blunt ends,
    2) overhanging 3 prime ends or
    3) overhanging 5 prime ends.
The protruding single-strands can be united with similar ends to readily produce recombinant molecules.

Isoschizomers are enzymes from different sources that target the same target sequence.
Depending upon how the cleavage sites compare, the enzymes may or may leave the same ends (compatible ends).
Often enzymes with a hexanucleotide recognition sequence (6-cutters) will leave a 4 nucleotide overhang.
Other enzymes that recognize a different hexanucleotide sequence that share the same central tetranucleotide motif may leave the same ends (compatible ends).

For example: SalI (GTCGAC) and XhoI (CTCGAG) leave compatible ends that once ligated, result in a site (GTCGAG) that is not cleaved by either enzyme.

Other  areas of concern when using Restriction enzymes
Double digestion conditions
Heat inactivation
Survival
Activity near DNA fragment ends
Star activity
Sensitivity to methylation

Restriction mapping
A segment of DNA can be characterized by breaking the molecule at defined sites.
The distance between the sites can calculated.
Map can be constructed.
 

Protocol

        Restriction Digest and Analysis of Plasmid

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