Restriction Enzymes

SOIL MICROBIOLOGY

BIOL/CSES 4684

Webpage created by John Thompson

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Model image of the restriction enzyme ECO RI (red and yellow) binding to DNA (blue). The enzyme is made up of two symmetrical units that correspond to the double helix structure of DNA.

1. Overview of Restriction Enzymes

Restriction enzymes, sometimes called restriction endonucleases, are short nucleotide sequences isolated from prokaryotic organisms that protect the cell from foreign DNA. Several hundred of these enzymes have been isolated from various organisms and most are available commercially. These enzymes recognize specific sequences, usually four to eight nucleotides in length. Different organisms produce enzymes with different sequence recognition sites, called restriction sites. Most organisms only recognize one specific site thus different enzymes will cut the same piece of DNA in different locations. Restriction enzymes cut the DNA strand by binding to the DNA at these specific sites and cleaving the double helix. The cut produces 3’-OH and 5’-P groups at each position. The cuts can either be straight or staggered. Straight cuts are referred to as "blunt" ends and staggered cuts are referred to as "sticky" ends. The piece of DNA that is produced from this cut is called a restriction fragment. There are three different types of restriction enzymes. They will de discussed in section 3. Some common restriction enzymes are EcoRI, HindIII, BamHI, and TaqI.

2. Overview of Procedure

Restriction enzymes are used for isolating and cutting a certain DNA sequence. The cut pieces of DNA can then cloned through a vector or placed into recombinant DNA. Since the majority of the isolated restriction enzymes are commercially available through biotech companies, the first step is figuring out which restriction enzyme to use. Almost all restriction enzymes have different restriction sites thus the choice of enzyme to purchase will also affect how the DNA sample will be cut. The enzyme treats the DNA sample by "scanning" the molecule. When it finds the restriction sequence, the enzyme binds to the sequence and cleaves the double helix. "Blunt" ends are harder to recombine than "sticky" ends. If the enzyme produces "sticky" ends, the DNA can be recombined relatively easily with DNA from another sample. "DNA fragments obtained from one organism will have the same sticky ends as the fragments from another organism if they have been produced by the same restriction enzyme" (Hartl, 353). Basically, most restriction enzymes recognize their restriction sequence no matter from where the DNA came. The following figure shows how the enzyme EcoRI enzyme cuts the DNA and the DNA then recombines:

Using an agarose gel that separates the DNA fragments can do detection of the recombinant DNA by size. Placing the fragment in a vector can also clone the fragment. Both of these practices are used extensively.

3. Pros and Cons

Restriction enzymes are very useful tools in cloning and recombinant DNA procedures. But with these useful tools come some good things and some bad things. One of the good characteristics is that in a relative sense, using restriction enzymes is an easy process and not very labor intensive. Since the enzymes can be purchased through biotech companies, that makes using the restriction enzymes easier because one doesn’t have to purify the enzyme from the microbe themselves which can be very time consuming. But the enzymes can also be very expensive to buy, which is a downside to using restriction enzymes. Another downside is having to decide which enzyme to use. The following table describes each type of enzymes characteristics:

Type II enzymes are the most widely studied and used enzymes because of their quality characteristics.

4. Sources

http://www.psc.edu/MetaCenter/MetaScience/Articles/Rosenberg/Rosenberg.html

http://www.gene.com/AE/AE/AEC/CC/action.html

http://www.gene.com/AE/AE/AEC/CC/restriction.html

http://www.bsi.vt.edu/molbiol/02-15-99.pdf

Hartl, Daniel L. 1994. Genetics. 3^rd edition. Jones and Bartlett Publishers, Boston. pp. 118-119, 352-354.

Alberts, B., D. Bray, J. Lewis, M. Raff, K. Roberts, J.D. Watson. 1994. Molecualr Biology of the Cell. 3^rd edition. Garland Publishing, New Yok. pp. 292-293.

5. Links to other sites on Restriction Enzymes

Promega: Supplies for the biotech industry. http://www.promega.com