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These are endonucleases which cleave double-stranded DNA molecules at specific sites having a sequence of generally four to six nucleotides. These sites are called restriction sites. A specific restriction enzyme cleaves DNA wherever the site occurs. The nucleotide sequence of the restriction site is specific for each of hundreds of restriction enzymes discovered so far from different microorganisms.
A restriction enzyme makes a cut in each of the two strands of DNA creating a free 3′-OH group and a free 5′-phosphate group. Majority of restriction enzymes cuts DNA strands within the restriction site, while some makes cuts outside it. The former group is useful for making recombinant DNA.
Restriction enzymes may cut DNA in two different ways, characteristic for a particular enzyme. Some cleave DNA strands, so that the cuts are opposite to each other producing blunt ends. Others produce cuts which are staggered, so that each cut piece of DNA has a short single-stranded end.
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The two types of cut caused by restriction enzymes are diagrammatically shown in Fig. 9.127:
Another characteristic feature of the restriction sites is that the base sequences of the sites are identical in both strands read from opposite directions. This type of symmetry is known as palindromic. For example, a very useful restriction enzyme, known as Eco R1 possesses a recognition sequence shown below.
Eco R1 produces staggered cuts of double-stranded DNA between G and A of the recognition site resulting in single-stranded ends which are complimentary to each other known as sticky ends as shown in Fig. 9.128:
The significance of restriction enzymes of Eco R1 type in recombinant DNA technology is that when the donor DNA and the vector DNA are both treated by the same enzyme, they produce complimentary sticky ends which may come together and form base pairs. If the open ends are then joined by DNA ligase, the two DNA molecules fuse to form a recombinant DNA molecule.
An important property of restriction enzymes is that they can cleave any DNA, irrespective of its origin, provided the DNA is double-stranded and possesses the restriction sites for the particular enzyme. The number of restriction sites of a particular enzyme depends on the size of the DNA molecule.
The larger the molecule, the higher is the probability of having larger number of sites for a particular enzyme. For example, the E. coli chromosome which is a very large DNA molecule possesses about 1,000 restriction sites for Eco R1, so that treatment with this enzyme produces about 1,000 fragments — each with sticky ends having the same sequence.
On the other hand, a smaller DNA molecule, like that of a plasmid, may have only one or two restriction sites for Eco R1. The plasmid pBR 322 has a single restriction site for EcoR1. Such plasmids are specially useful as cloning vectors.
Characteristics of some restriction enzymes are shown in Table 9.8:
Although the restriction enzymes producing sticky ends are more useful for cutting DNA and joining them to construct recombinant DNA, sometimes sites of such enzymes may be lacking in the DNA segments. In such cases, the enzymes producing blunt ends may have to be used, provided there are sites in the DNA segments to be treated.
For making the blunt end DNA produced by these enzymes usable, a synthetically produced short DNA segment containing a restriction site of Eco R1 is joined at both blunt ends. These short sequences are called linkers. The blunt ends then produce sticky ends by treating the DNA with Eco R1.
The procedure is diagrammatically shown in Fig. 9.129:
Such DNA segments containing sticky ends can then be joined to a plasmid DNA which has also been cut with Eco R1. Then the usual procedure is followed for constructing a recombinant DNA molecule. The general procedure for obtaining a recombinant DNA by joining a DNA segment containing sticky-ends to a plasmid DNA having a single site for a restriction enzyme, like Eco R1 is illustrated in Fig. 9.130.
