This is a replay of our biotechnology training workshop which was held on April 29 at 3:30 PM EDT.

Restriction enzymes are powerful tools in the biotechnology laboratory. They allow researchers to cut DNA in precise locations based on its sequence. This is important for molecular cloning and DNA fingerprinting analysis. But did you know that they are easy to use in your laboratory? In this live stream, we demonstrate the use of restriction enzymes in your classroom with our best selling experiment, "DNA Fingerprinting Using Restriction Enzymes." We discussed the history of restriction enzymes in the laboratory and tips and tricks to make your experiment a success.

For the experiment: [ Ссылка ]
For the slides: [ Ссылка ]
For our past discussion of DNA Fingerprinting: [ Ссылка ]
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One of the most significant discoveries of molecular biology is a class of enzymes that can slice DNA in a specific and reproducible manner. Restriction endonucleases target the double-stranded DNA at specific sequences and break through the DNA backbone. This fragments the DNA into smaller pieces.

Digestion of the same piece of DNA with different enzymes will create unique patterns. And the relationship between these cut sites, the order, the number, and more let us characterize a gene or a piece of the genome without knowing its sequence. And this was very important before DNA sequencing became more inexpensive and readily available.

Much like CRISPR, restriction endonucleases evolved in bacteria as a defense against viral attacks. So, here we see bacteriophage virions attached to the outside of a bacterial cell, and in some we can actually see the genetic material being injected into the cell. As the invading viral DNA enters the cell, the enzymes recognize that the DNA is foreign based on specific modifications that have been made to the DNA. At this point, it chops the invader’s DNA into pieces to prevent it from within the bacterial cell, which would generally end in cell lysis.

There are five types of restriction enzymes that have been isolated from bacteria, but Type II are most common in the laboratory. These enzymes bind to the DNA as homodimers, as seen here. This is a complex where two of the same proteins bind to one another. Each protein has several domains, including a DNA binding domain which binds with the recognition site, and an endonuclease domain that cleaves the DNA. Most of these enzymes require Mg2+ as a cofactor, meaning that we have to be careful to use the correct buffer for best results.

Restriction enzymes can either cleave the recognition site at the center of the DNA strands to yield a blunt end, or at a staggered position leaving overhangs called sticky ends. To illustrate this, first consider the recognition site and cleavage pattern of HaeIII. This enzyme cuts both DNA strands at the same position, which generates fragments without an overhang. These so-called “blunt” ends can be joined with any other blunt end. In contrast to HaeIII, enzymes like EcoRI cleave are in a staggered form, so the resulting fragments project short overhangs of single-stranded DNA with complementary sequences. Such overhangs are referred to as “sticky” ends because the single strands can interact with—or stick to—other overhangs with a complementary sequence.

If we are cloning these genes into vectors, we would use these sticky ends for specificity in cloning, but we’re going to this sequence specificity for our analysis. Digestion of the same piece of DNA with different enzymes will create unique patterns that can tell us about their sequence, and this is the principle behind DNA fingerprinting.

The probability that a given enzyme will cut, or “digest”, a piece of DNA is directly proportional to the length of its recognition site. Statistically, an enzyme will average one cut for every 4 to the n base pairs, where n is the length of the recognition site. For instance, an enzyme that recognizes a four base pairs long sequence (e.g., HaeIII) will cut DNA once every 256 base pairs, while an enzyme that recognizes a six base pairs long site (e.g., EcoRI) will cut once every 4096 base pairs. Therefore, the longer a DNA molecule is, the greater the probability is that it contains one or more restriction sites. For example, if EcoRI is used to digest human chromosomal DNA containing 3 billion base pairs and a plasmid containing 5,000 base pairs, it will cut the chromosomal DNA over 700,000 times (3 billion base pairs, cut every 4096 base pairs), but may only cut the plasmid once (5,000 base pairs, cut every 4096 base pairs).

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