What are restriction enzymes?

Restriction enzymes, or restriction endonucleases, are proteins produced by bacteria to defend against foreign DNA by cutting it into nonfunctional pieces. These enzymes act as DNA scissors, specifically recognizing and cleaving DNA at restriction sites. A nuclease is any enzyme that cleaves the DNA backbone by breaking phosphodiester bonds. Endonucleases cut within a DNA molecule at specific sequences, while exonucleases cut from a free DNA end. Exonucleases typically act on single-stranded DNA (ssDNA) or nicks in double-stranded DNA (dsDNA), whereas endonucleases act exclusively on intact dsDNA.

Restriction enzymes are notable for their ability to distinguish between native and foreign DNA, such as bacteriophage DNA, and are critical in cellular defense mechanisms. Restriction sites are specific 4- or 6-base-pair palindromic sequences, where the 5′-to-3′ sequence on one strand matches the 5′-to-3′ sequence on the complementary strand. For instance, the sequence 5′ GAATTC 3′ is a palindrome and serves as the restriction site for EcoRI, a restriction enzyme derived from Escherichia coli RY13.

EcoRI cuts the DNA between the G and A in each strand, creating single-stranded “sticky ends” that can easily rejoin with complementary sticky ends. Not all restriction enzymes produce sticky ends; some, like SmaI, generate “blunt ends” by cutting both strands at the same position. Restriction enzymes widely used in laboratories typically recognize specific palindromic sequences and cleave the DNA at precise positions within these sites.

The cleavage pattern of restriction enzymes results in DNA fragments whose sizes and numbers depend on the locations of restriction sites within the DNA molecule. For instance, sequences of 4 bases occur randomly approximately once every few hundred bases, while 6-base sequences occur less frequently.

Under suitable conditions of salt concentration, pH, and temperature, a restriction enzyme can produce consistent DNA fragment patterns. Some DNA molecules, such as bacteriophage lambda (~48,000 base pairs), have diverse restriction sites, while others may lack specific restriction sites altogether. Restriction enzymes are indispensable tools in molecular biology for DNA analysis, cloning, and other applications.

What are some applications for restriction enzymes?

The discovery of restriction enzymes made genetic engineering possible because restriction enzymes first made it possible to work with small, defined pieces of DNA. Chromosomes are huge molecules that usually contain many genes. Before restriction enzymes were discovered, a scientist might be able to tell that a chromosome contained a gene for an enzyme required to ferment lactose because he knew that the bacterium could ferment lactose, and he could purify the protein from bacterial cells. He could use genetic analysis to tell what other genes were close to this gene. But he could neither physically locate the gene on the chromosome nor manipulate that gene. The scientist could purify the chromosome from the bacterium, but then he had a huge piece of DNA containing thousands of genes. The only way to break the chromosome into smaller segments was to use physical force and break it randomly. Then what would he have? A tube full of random fragments. Could they be cloned? Not by themselves. If you introduce a simple linear fragment of DNA (like those produced by shearing) into most bacteria, it will rapidly he degraded by cellular nucleases. Cloning usually requires a vector to introduce and maintain the new DNA. Could our scientist use a vector such as a virus or plasmid to clone his DNA fragments? No. In order to clone DNA into a vector, you have to cut the vector DNA to insert the new piece. Could he simply study the random fragments? No. Every single chromosome from each bacterial cell would give different fragments, preventing systematic analysis. So, for many years, physical manipulation of DNA was virtually impossible.

The discovery of restriction enzymes gave scientists a way to cut DNA into defined pieces. Every time a given piece of DNA was cut with a given enzyme, the same fragments were produced. These defined pieces could be put back together in new ways. A new phrase was coined to describe a DNA molecule that had been assembled from different starting molecules: recombinant DNA. The seemingly simple achievement of cutting DNA molecules in a reproducible way opened a whole new world of experimental possibilities. Now scientists could study specific small regions of chromosomes, clone segments of DNA into plasmids and viruses, and otherwise manipulate specific pieces of DNA.

Let’s cut a viral genome with restriction enzymes, on paper: In silico lambda DNA digestion (adapted from The American Phytopathological Society APS).

In silico = in or on a computer, done or produced by using computer software or simulation. In other words, you’ll practice on paper an enzymatic restriction digestion before you do it on DNA.

1. Download and SAVE the Lambda DNA sequence (lambdafasta).
2. Save the file on your computer.
3. Open the file in Microsoft Word. It contains the entire phage Lambda DNA genome.
4. Determine how many letters there are in the genome. This number correspond to the number of bases of genome size in bp.

Simulating the Effects of Restriction Enzymes

Remember that there are a large number of restriction endonucleases (restriction enzymes), and that each enzyme recognizes a specific sequence of DNA nucleotides and cuts at a specific point within that sequence. The four restriction enzymes we will use for this exercise are BamHI, EcoRI, HindIII and AluI.

For each enzyme perform a “single digest” simulation and count the number and size of fragments produced using the following procedure:

1. Position the cursor on the first letter of your sequence (the first line starting with the symbol “>” is the name of the sequence).
2. Using the SEARCH option available on your operating system look for the motif that correspond to each enzyme’s restriction site (e.g. GAATTC). The number of hits corresponds to the number of restriction sites within your complete sequence.
3. Position the cursor after the first letter of the restriction site and select the text “previous” to it. Using the word count option of your text editor obtain the number of characters from the beginning of the sequence. This number will give you the first position of the restriction site and the size of the fragment.
4. Repeat for each restriction enzyme.
5. Indicate the fragment sizes, from small to large, on the following map:

Now simulate a gel electrophoresis where you load each of the four lanes of the gel with a different sample of your restriction enzyme digests. “Turn on” the power supply and watch your DNA move down the gel. Draw the bands of DNA fragments where they would settle on the gel, according to size order, using the DNA ladder lane as a guide.

Materials

• Four Eppendorf microtubes
• Microtube rack
• Micropipette set
• Beaker or foam cup with crushed ice for the following
  • o 20 µl of 0.4 µg/µl λ DNA
  • o 5 µl BamHI restriction enzyme
  • o 5 µl EcoRI restriction enzyme
  • o 5 µl HindIII restriction enzyme
• Restriction enzymes buffers
• 10 µl distilled water
• Electrophoresis chamber
• Power supply
• 20 µl 10X loading buffer
• 1.0% agarose gel

Safety concerns

This experiment uses chemicals, biological materials, and equipment  that pose some potential risks:

1. Handle the ethanol and the kit reagents with gloves, at all times.
2. Use biosafety protocols to handle the DNA samples  to prevent accidental exposure, and dispose of all the waste in a biohazard container.
3. Make sure you balance the  microcentrifuges correctly before you use them.
4. Avoid eating or drinking, and manage spills and emergencies following the directions of the instructor.

Protocol

1. Put on gloves. Keep all enzymes and DNA aliquots on ice through step 6.
2. Label 2 PCR tubes with the name of the enzymes indicated below, and place them in the tube rack.
3. Spin down the tubes before opening them to ensure the liquid is collected at the bottom.
4. Add each reagent in the order indicated, making sure to transfer the complete volume.
5. Follow the directions given by your instructor.
6. Close the microtubes, mix the content by flicking the tubes, spin the tubes down and place them on ice.
7. Incubate the tubes following your instructor’s directions.
8. Freeze the digested samples or run on a 1% agarose gel along a 1Kb DNA ladder.

Lecture slides

Download the slides to follow the pre-lab lecture. enyzmaticdigestion_2025

Lab report

Download the EnzymaticRestrictionLabReport file and submit on Canvas, following the directions of your instructor.