How Restriction Enzymes are Used to Cut DNA

What is covered in this Lesson

    1. Restriction Enzymes
      1. Where do they come from?
      2. What do they do?
      3. Applications in technology
      4. Review 
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By the end of this lesson you should be able to:

    1. Compare the natural role of restriction enzymes in a bacteria versus the application of restriction enzymes for biotechnology.
    2. Demonstrate how restriction enzymes recognize and separate strands of DNA.

All About Restriction Enzymes

What are they?

Bacteria naturally produce restriction enzymes (also called restriction endonucleases) as a defense mechanism against foreign DNA. Just like us, bacteria can be infected by viruses. However, the viruses that are specific to bacteria and are called bacteriopahges. When a bacteriophage attempts to infect a bacterium, it injects its genetic material into the cell. In an attempt to defend itself, the restriction enzymes within the bacterium recognize specific DNA sequences that are unique to the viral DNA and cut them into pieces. Now that the foreign DNA has been chopped up, it is no longer able to harm the bacterium. To see what this looks like in action, watch the video in the review section.

 


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How do they work?

In part A, the figure shows a strand of ladder-like DNA. In part B, the DNA is cut on both strands between the two guanines. In part C, the 2 strands have separated, leaving complementary sticky ends on each with unattached 5' to 3' G, A, T, and C nucleotides.

All restriction enzymes recognize a sequence of four- to eight-nucleotides called the recognition site. The nucleotide sequence is palindromic, meaning the sequence of ATCG’s read the same forwards on one strand and ‘backwards’ on the complementary strand. Look at the image to the right, do you recognize the palindrome? You’ll notice they read the same in the 5′ to 3′ direction on one strand as they do in the 5′ to 3′ direction on the complementary strand (a).
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On that image, you’ll notice the six-nucleotide recognition sequence (GGATCC) for the restriction enzyme BamHI.  Restriction enzymes not only recognize specific sequences, . In the case of BamHI, the cut is made between the two guanines like this: G | GATCC. It is important to note that the cut is made between the two guanines for both DNA strands. This might be a little confusing since the strands go in opposite directions, but if the enzyme cuts G | GATCC on one strand, it also cuts G | GATCC on the other strand.

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Many restriction enzymes make the staggered cuts you can see in the image (c); where each strand of DNA  is left with a short nucleotide single-stranded overhang called a “sticky end”. We call these overhangs “sticky ends” because they’re capable of coming back together through hydrogen bonding.

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How can we use them?

If you were cloning a gene, another piece of DNA – cut with the same restriction enzyme – could attach to these sticky ends and be inserted into the space made by this cut. If we want to break down the DNA to analyze their fragment sizes, we can add both the DNA and the restriction enzyme to a solution in a test tube. Under the right conditions and after a length of time, the restriction enzymes will act as a scissor to cut the DNA at its respective recognition sequence yielding DNA fragments of various lengths. If we want to further analyze these fragments, we can then use agarose gel electrophoresis to separate the DNA fragments by their difference sizes.

Restriction Enzyme Review Questions

Conclusion Questions

  1. Where are restriction enzymes found in nature?
  2. How do restriction enzymes protect bacteria from being harmed?
  3. Can restriction enzymes be purified and used in other applications?
  4. How do restriction enzymes know where to cut DNA?
  5. What is the name for the ‘flaps’ of DNA that are left after a restriction enzyme has cut it?
  6. How are DNA fragments processed and analyzed in a gel electrophoresis experiment?

Key Words

  • Bacteriophage
  • Restriction Endonuclease
  • Sticky-ends
  • Recognition Site

Course Navigation

References

  1. Fowler, Samantha, Rebecca Roush, and James Wise. “Molecular Biology.” Concepts of Biology. Houston, TX: OpenStax, 0. https://openstax.org/books/concepts-biology/pages/10-1-cloning-and-genetic-engineering. License: CC BY 4.0 License Terms: Edited & Adapted | Access for free at https://openstax.org/books/concepts-biology/pages/1-introduction
  2. Helixitta. “File:HindIII Restriction site and sticky ends vector.svg.” Wikimedia Commons. 29 July 2015. ​https://commons.wikimedia.org/wiki/File:HindIII_Restriction_site_and_sticky_ends_vector.svg​License: CC BY 4.0 License Terms: No edits were made
  3. Ancient Lights. “How Do Restriction Enzymes Work?.” Discovery Education, 2014. app.discoveryeducation.com/learn/videos/1ccdfe69-eb7c-4e31-bba8-92de5aaf8827.
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