How are restriction enzymes and DNA ligase used to combine foreign DNA with a plasmid?

BIOL 1406

PreLab 9a.3

How do restriction enzymes cut DNA into fragments and how can these fragments be analyzed using agarose gel electrophoresis?

Ribbon diagram of the restriction enzyme EcoRI

Restriction enzymes are naturally occurring enzymes that cut DNA. Many restriction enzymes have been isolated from bacteria, providing a valuable tool for molecular biologists. Each restriction enzyme cuts DNA only where a specific sequence of base pairs occurs. For example, these are the sequences cut by the restriction enzymes BamHI and XmaI.:

An important difference among restriction enzymes is whether the cuts they make produce “blunt ends” or “sticky ends.” For example, HaeIII cuts this sequence:

Because these 2 fragments do not have any unpaired bases, we say they have “blunt ends.” EcoRI, on the other hand, cuts this sequence:

Because these 2 fragments have unpaired bases which can potentially pair with complementary bases, we say they have “sticky ends.”

If we cut DNA from two different sources with the same restriction enzyme, DNA fragments with complementary sticky ends are formed:

If the DNA fragments are mixed together, they will join together at random, provided their unpaired bases match. Therefore, sometimes pieces of DNA from the same source will join back together, and sometimes pieces of DNA from different sources will join together, as shown below.

Hydrogen-bonding between complementary bases is not sufficient to completely rejoin the DNA fragments. The broken bonds between the deoxyribose and phosphate groups that form the “side-rails” of the DNA double helix (the phosphodiester linkages) must also be repaired. DNA ligase is the enzyme, also isolated from bacteria, that catalyzes this reaction.

In this lab exercise, you will be given 3 unknown samples labeled A, B, and C. One sample has no plasmids at all, one sample has normal pUC18 plasmids, and one sample has recombinant pUC18 plasmids. Your job is to determine which plasmids, if any, are present in each sample. To do this, you will carry out 2 experiments:

E. coli cells will be mixed with each of the 3 unknown samples in order to allow them to absorb any plasmids that are present. The E. coli cells will then be plated on nutrient agar containing ampicillin and Xgal in order to determine what type of colonies, if any, develop.
The restriction enzyme EcoRI will be mixed with each of the 3 unknown samples in order to cut (or digest) any plasmids that are present. The resulting DNA fragments will then be separated using agarose gel electrophoresis in order to determine what size fragments, if any, were produced.

In this lab exercise, you will be using agarose gel electrophoresis to separate the DNA fragments that were produced during the EcoRI digest of your 3 unknown samples (A, B, and C.) Click the play arrow on the left to watch a short video about how to prepare, load, and run your agarose gel.

If you have problems seeing the video, you can view it in an External Viewer

Your Turn
What is a recombinant plasmid?	Check your answer.
What are restriction enzymes?	Check your answer.
What is DNA ligase?	Check your answer.
If a pUC18 plasmid is cut in only one location by the restriction enzyme EcoRI, it will be changed from a circular form to a form.	Check your answer.
Imagine that both pUC18 plasmids and phage DNA are cut with EcoRI. Each plasmid is cut in one location to form a linear fragment and each phage is cut in one location to form a linear fragment. Then all of the linear fragments are mixed together. In the spaces below, list the five possible ways circular pieces of DNA can form by joining the ends of either one or two of these linear DNA fragments. Note that the 2 ends of one fragment can join to form a circle, or two different fragments can join to make a circle: 1. 2. 3. 4. 5.	Check your answer.
Which of the 5 possible combinations represent a recombinant plasmid?	Check your answer.
Which of the 5 possible combinations represent plasmids that are not recombinant?	Check your answer.
Which of the 5 possible combinations represent circular pieces of DNA that do not include a plasmid?	Check your answer.
After cutting recombinant and non-recombinant plasmids into fragments with the restriction enzyme EcoRI, which procedure will you use to separate the fragments and determine their size?	Check your answer.

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