Molecular Biology: Gene cloning
Molecular Biology:
Gene cloning
Author: Prof Marinda Oosthuizen
Licensed under a Creative Commons Attribution license.
RESTRICTION ENDONUCLEASES
To incorporate fragments of foreign DNA into a cloning vector, methods for cutting and rejoining of
single stranded DNA are necessary. The identification of restriction endonucleases in the 1960s and
early 1970s and the recognition that these enzymes act as “molecular scissors”, always cutting DNA
at specific locations (base sequences), was the key discovery which allowed the cloning of DNA to
become a reality (Turner et al., 1997). Restriction enzymes are proteins that bind to a specific DNA
sequence and cleave (hydrolyze) the backbone of the DNA between the deoxyribose and phosphate
groups (the phosphodiester linkage). This results in phosphate groups on the 5’ ends and hydroxyl
groups on the 3’ ends of both strands. The biological function of restriction enzymes is to protect the
bacterial cell against the introduction of foreign DNA into the cell (Turner et al., 1997). DNA
methylases also exist in the cell to prevent the restriction enzymes from digesting the cell’s own DNA.
This is done by the modification of the DNA at specific sites which prevents the restriction enzymes
from binding or cleaving the DNA.
Restriction enzymes are distinguished according to their recognition sequence and their cleavage
site. Three main groups have been recognized:

Type I enzymes that cut DNA up to 5 kbp away from the recognition site,


Type II enzymes which cut DNA inside the recognition site, and
Type III that cut DNA just a few base pairs away from the recognition site (also called shifters).
In recombinant DNA technology, the lengths of the fragments generated, and the composition of the
termini of the fragments generated are the two important criteria to consider in the choice of a
restriction enzyme. The length of the restriction fragments generated will depend on several factors,
including (i) the length of the recognition sequence and its composition, (ii) the G+C content of the
organism’s DNA, and (iii) its methylation status (most restriction enzymes do not recognize
methylated palindromes and do not cleave these sequences). With regards to termini generated upon
restriction, three types are differentiated:

5’-extensions (5’-sticky or cohesive ends) as generated by most restriction enzymes, eg. EcoRI


(G’AATTC)
3’-extensions (3’-sticky or cohesive ends) as generated by others such as PstI (CTGCA’G)
Blunt or flush ends produced for example by PvuII (CAG’CTG) or HaeII (GG’CC).
These features are crucial and have to be taken into consideration when manipulating DNA since
nucleases and polymerases recognize certain types of ends only. When joining (ligating) together
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Molecular Biology: Gene cloning
restriction fragments, it is important to have complementary sticky ends, which facilitate the annealing
process
and,
therefore,
increase
(http://www.brunel.ac.uk/depts/bl/project/genome/moltec/).
ligation
efficiency
Restriction enzymes as “molecular scissors”:
(http://www.hort.purdue.edu/hort/courses/HORT250/lecture%2003)
1. Restriction enzymes recognize a specific sequence of bases in a DNA molecule. In the
example shown below, the enzyme EcoRI finds the sequence G’AATTC.
2. The restriction enzyme then cuts through the two strands of DNA molecule in a very precise
manner, indicated by the red line. Fragments of DNA produced by restriction enzymes can now be
used for cloning. Note that this enzyme has cut the DNA to leave protruding sticky ends.
3. Some other restriction enzymes cut DNA to produce blunt ends, for example HaeII:
A restriction digest will result in a number of DNA fragments, the sizes of which depend on the exact
positions of the recognition sequences for the endonuclease in the original DNA molecule. These
DNA fragments often require separation and/or analysis. Gel electrophoresis is frequently used to
separate fragments of DNA (or RNA) according to their size and is commonly used to identify specific
cloned genes.
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