Virtual digests & restriction maps

This restriction digest lab is about cutting plasmid DNA with restriction enzymes as a way to help differentiate the various plasmids you're working with. There are two parts to this lab:

  • A virtual digest, in which you use a computer and some paper to analyze DNA sequences and predict the results of restriction digests. This should help you to interpret the results of your real restriction digests. Do this exercise in lab with your group and turn it in today.
  • Real restriction digest, in which you cut your purified plasmid DNA with restriction enzymes in one lab period and analyze your digested DNA using electrophoresis in the next lab period.

You're working on two concurrent lab projects involving plasmids: conjugation with pARO180 and transformation with pGLO. So far, if all has gone well, you've used two different mechanisms for getting E. coli cells to take up plasmids, and you have seen plasmid DNA on total nucleic acid gels. However, there are a couple of problems with visualizing the plasmid DNA on a total nucleic acid gel along with chromosomal DNA and some RNA. First, that method doesn't yield much plasmid DNA, and you might not have seen it at all. Second, it doesn't allow you to tell the various plasmids apart, since they're all around the same size. You should be able to solve both those problems with a two-step approach:

  • Purifying plasmid DNA (which you've already done) should give you a greater yield, and eliminate everything but plasmid DNA.
  • Cutting the purified plasmid DNA with restriction enzymes should allow you to distinguish the plasmids. Each plasmid has a different nucleotide sequence, and you can set up your experiment so that each plasmid will get cut into DNA fragments of a characteristic size. Finally, you'll analyze the size of the fragments on a gel.

Before you start cutting the DNA, you need to analyze the sequences to figure out where the enzymes will cut each plasmid. In the computer-based exercise on this page, you'll explore the connection between nucleotide sequences, restriction enzymes, and DNA fragment sizes by using sequence analysis software to figure out your expected results from plasmid digests.

This exercise is a first foray into bioinformatics: using software tools to analyze biological data such as nucleotide sequences. You'll continue this approach in the open reading frames assignment.

Background

Restriction enzymes cut DNA

Restriction enzymes are proteins that cut DNA at specific sites, determined by the nucleotide sequence of the DNA. Like most of the tools in a molecular biologist's toolkit, restriction enzymes are taken from cells. Cells do all sorts of tricks with DNA; learning how to manipulate DNA in a test tube usually means mimicking the way DNA is manipulated inside a cell. Cells manipulate DNA by using enzymes: proteins that catalyze specific chemical reactions. Molecular biologists get enzymes from cells, then use the enzymes in new ways.

Restriction enzymes cut DNA at specific sites. They recognize short sequences of nucleotides, called restriction sites, and cut the DNA at those sites. Restriction sites are usually four to eight base pairs long. There are many different restriction enzymes, each with its own specific restriction site. Here is the restriction site for Hind III, one of the restriction enzymes used in the 6B lab:

Hind III restriction site

In this diagram, N refers to any nucleotide. The Hind III protein bounces along the DNA until it recognizes its specific restriction site, AAGCTT. Once it finds that sequence, it cuts each strand of the DNA, breaking the covalent bonds that join the nucleotides. In this case, the two strands are not cut at the same location, so each resulting DNA fragment has a 4-nucleotide overhanging "sticky end."

If there are multiple Hind III restriction sites in a DNA molecule, the enzyme will cut all of them. Thus, a restriction digest tells us something about a DNA sequence. If you start with a single linear fragment of DNA and cut it with Hind III and then discover that it has been cut into three smaller fragments, you have learned that the original DNA fragment contained two restriction sites for that enzyme. You'll be able to compare different DNA molecules by testing how each gets cut with specific restriction enzymes.

Restriction enzymes are also called endonucleases, because they cut nucleic acids somewhere in the midst of the molecule (endo- means in). Exonucleases also exist; they cut up DNA by removing nucleotides from the ends of the molecules, rather than cutting at specific sites. (The enzyme DNA polymerase I acts as an exonuclease when it eats away the 5' end of a primer during DNA replication.)

Restriction enzymes were the first DNA-altering enzymes to be isolated and used in the laboratory; producing recombinant DNA by cutting and joining DNA fragments was one of the foundational techniques for modern molecular biology.

Bacterial cells use restriction enzymes for self-defense

Restriction enzymes cut up DNA. You might think that this would be bad for a cell; normally, cells don't cut up their own DNA. However, sometimes it's beneficial to cut up DNA that doesn't belong inside a cell. The restriction enzymes you'll use come from bacteria, and the bacteria use the enzymes to cut up DNA from viruses or other potentially dangerous sources. So the restriction enzymes function as a self-defense weapon for the cell.

Bacteria commonly protect their own DNA against restriction enzyme by methylating (adding methyl groups to) the DNA at the restriction sites.

Cutting DNA is useful in the lab

In this lab, you'll use restriction digest to allow you to distinguish various plasmids with similar sizes but slightly different DNA sequences. By using restriction enzymes that target the differences between the plasmids, you should be able to get a unique set of restriction fragments for each plasmid.

Restriction digests also form part of the foundation for molecular biology in general. Recombinant DNA molecules (such as the pGLO plasmid) are traditionally made by cutting various desired DNA fragments with restriction enzymes, then ligating (joining) the pieces together.

The restriction enzymes for this lab

In this lab, you'll have three different restriction enzymes to cut your plasmid (and virus) DNA. The restriction enzymes are:

Bio 6B restriction enzymes, showing restriction sites

The first three (Hind III, Pvu I, and Ssp I) are 6-base cutters; their restriction sites are 6 nucleotides long. Note that Hind III and Pvu I leave sticky ends, while Ssp I leaves blunt ends: both strands are cut at the same place.

Sat I is different from the others. First, its restriction site is only five nucleotides long. Also, in this restriction site, "N" in the middle means that any nucleotide will work; as long as Sat I finds the other nucleotides in the sequence, it will cut the DNA. This means that Sat I is likely to cut most DNA sequences at many locations. You wouldn't use it to cut your plasmid DNA, because it would cut the DNA into too many small fragments. However, it's useful in the PTC PCR lab, because it cuts the short PCR product from that lab at only one site.

Why you're cutting plasmids

As mentioned earlier, you're going to cut your plasmids so you can tell them apart. Plasmids normally exist as circular, double-stranded DNA molecules in bacterial cells. In addition to the double helix secondary structure found in almost all DNA, these circular DNA molecules can wind around themselves to form tightly coiled, compact supercoiled conformations. Thus, this type of DNA has tertiary structure. Supercoiled DNA, due to its compact shape, runs faster on a gel compared to DNA that is not supercoiled. When you compare your supercoiled plasmid DNA to a linear molecular weight marker (as you did on the total nucleic acid gel), you don't get a true indication of the size of the plasmid. This can make it difficult to know whether you are seeing the plasmid that you expect.

Keep in mind that there are usually multiple copies of the plasmid in a single cell. The plasmids that we are using, pARO180 and pGLO, are high copy number plasmids; there are usually hundreds of copies of the plasmid DNA per cell. Even if most of the plasmid DNA is supercoiled, a small percentage of it might be in the linear (if both strands got broken) or open circle (if one DNA strand got broken, allowing the supercoiling to untwist). Thus, you could potentially have several bands of plasmid DNA on a gel, even if all the plasmid molecules are the same length and same molecular mass.

Cutting circular DNA with a restriction enzyme that cuts at only one site will linearize the DNA (convert it from circular, supercoiled form to linear form). This allows for more precise determination of the size of the DNA fragments. In this lab experiment, you will start with two different plasmids that are close to the same size (pGLO is 5371 base pairs long, and pARO180 is 5460 bp). When they are supercoiled, it might be difficult to tell them apart on a gel. Each of these plasmids has one restriction site for Eco RI, so this enzyme will linearize them, making it easier to tell them apart.

You can get more information by using restriction enzymes that cut more than once. pGLO has two Hind III restriction sites, while pARO180 has only one. If you cut the plasmids with Hind III, pGLO produces two bands on the gel, but pARO180 only one. That's a good way to tell these plasmids apart.

Different versions of pGLO

You should have three different versions of pGLO:

pGLO Green:
The standard, unmutated version of pGLO, which contains the gene for Green Fluorescent Protein (GFP).
pGLO Blue:
Mutated version of pGLO, which contains a gene for Blue Fluorescent Protein (BFP). The nucleotide sequence of this plasmid contains a couple of nucleotide substitutions compared to regular pGLO, but these differences do not affect any restriction sites, so the digested plasmid DNA should look the same as the unmutated version.
pGLO Non-F:
Mutated version of pGLO. In this case, the mutation makes the GFP gene non-functional, so the colonies are non-fluorescent. The plasmid still gets replicated in the cells, and it still contains the ampicillin resistance gene, so you should get non-fluorescent, amp-resistant colonies with this one. The mutation that disrupts the function of the GFP gene also adds a new SspI restriction site to the plasmid, so this mutated version should give a different set of bands on the gel compared to the pGLO and pGLO Blue.

Use λ bacteriophage DNA as a marker

In addition to the plasmids, you'll also cut some DNA is from a virus called lambda (abbreviated with the Greek letter λ). It's a bacteriophage, meaning it's a virus that infects bacteria. The genome of λ bacteriophage is linear, not circular, and it's about 43,000 bp long, or 43 kb. When λ DNA is cut with Hind III, it produces a recognizable and predictable set of bands that can serve as a molecular weight marker. Some of the bands are close in size to the plasmid DNA, so λ/Hind III is a good marker for this experiment.

Restriction map & virtual digest

DNA maps are simplified representations of nucleotide sequences, showing features of particular interest such as genes. Restriction maps are DNA maps that show restriction sites, the locations where specific restriction enzymes will cut the DNA. Here is a map of pGLO:

pGLO restriction map

I made this map with Benchling, the online software package that I usually use for analyzing nucleotide sequences. Some of the features on this pGLO map should look familiar, such as the ampicillin resistance gene (AmpR) and the GFP gene. Also included are restriction sites for three enzymes that we have in lab: HindIII, PvuI, and EcoRI. This map will give you a general idea of what size DNA fragments to expect if you cut pGLO with each of these enzymes:

  • PvuI: Cuts once, so you get one linear piece, the size of the entire plasmid (5371 bp).
  • EcoRI: Cuts once, so you get one linear piece, the size of the entire plasmid (5371 bp). Although EcoRI cuts in a different place than PvuI, the resulting fragment will be the same size.
  • HindIII: Cuts twice, so you will get a small piece and a large piece.

Now suppose you want to cut the plasmid with more than one enzyme at a time. Each enzyme will act on its own restriction sites, so the different enzymes won't affect each other. From the map above, you can see that if you cut with both EcoRI and HindIII, you will cut out a tiny piece between the EcoRI and HindIII sites; you probably wouldn't be able to see this small fragment on a gel, so it's not worthwhile to perform this digest. Suppose you cut with PvuI and EcoRI together. Using this slightly more detailed map (courtesy of Snapgene), you should be able to predict the exact sizes of the DNA fragments you will get:

pGLO snapgene

This map shows the exact locations of the restriction sites. Each nucleotide is assigned a number, based on an arbitrarily chosen starting point. If PvuI cuts at nucleotide 3054 and EcoRI cuts at nucleotide 2063, and the entire circular plasmid is 5371 bp long, you can probably figure out the sizes of the fragments you will get.

Unfortunately, the map above leaves out the HindIII sites (I don't know why they weren't included).

Perform some virtual digests of pGLO

Using the restriction enzymes we have in lab, the best combination for getting nicely spaced fragments of pGLO on your gel would be HindIII and PvuI. To figure out the expected results of the HindIII + PvuI digest, you can use restriction mapping software such as this one:

Restriction Analyzer from molbiotools.com.

For a precise electronic restriction digest, you need to enter the nucleotide sequence of the DNA you're going to cut. Find the pGLO sequence (the original sequence from Bio-Rad) on the Sequence Data page (keep that page open in another tab; you'll use it again).

Copy the whole sequence, including the numbers, and paste it into the "sequence info" box in the Restriction Analyzer (the software will ignore the numbers and spaces, and only look at the nucleotide sequence). Note that the sequence is shown for only one strand of DNA, but the software is smart enough to recognize that the actual DNA is double-stranded. Check  "Circular" under Conformation, and then go to the Include box and select PvuI. Click on Virtual Digest.

Virtual digest will take you to a page showing the complete nucleotide sequences of the DNA fragments produced in the digest (which you probably don't care about) and also the length of each fragment (which you will be able to verify on your gel. Those fragment lengths are your expected results for this experiment.

Repeat the virtual digest procedure for the enzyme SspI.

Alternative approach: You could also use Benchling instead of RestrictionMapper. Benchling is more modern and more powerful, but it takes a little while to learn and you will have to sign up for a (free) account. Personally, I use Benchling for most of my sequence work, and I recommend it.

Make your own map of pGLO

Draw your own simplified map of pGLO. Show the exact cut sites for PvuI and SspI. Also include the approximate locations of the GFP, AmpR (ampicillin resistance, also called β-lactamase or bla), and AraC genes.

Perform virtual digests of pGLO non-fluorescent mutant

The  Bio 6B Special Projects students accidentally created a mutant version of pGLO that doesn't have a functional GFP gene. Find the sequence for the pGLO Non-Fluorescent Mutant on the sequence page.

Perform virtual digests of this sequence the same way you did with the original pGLO sequence. You should get a slightly different result for one of the enzymes.

On the pGLO map, show where the mutant sequence differs from the original.

Perform a virtual digest of lambda

Lambda bacteriophage is the DNA you'll use as a molecular weight marker. This DNA is widely available gets cut into a range of predictably sized fragments, making it one of the most common molecular weight markers.

Find the sequence of lambda on the Sequence Data page. The lambda genome is linear DNA, unlike the circular plasmids.

Perform a virtual digest of lambda with the enzyme Hind III and write down the fragment sizes. Unlike the plasmids, lambda DNA is linear, so check the appropriate box before doing the digest.

Diagram the gel results

Draw a labeled picture of the expected gel results for the following restriction digests:

  • pGLO Green cut with PvuI
  • pGLO Green cut with SspI
  • pGLO Non-Fluorescent cut with PvuI
  • pGLO Non-Fluorescent cut with SspI
  • Lambda DNA cut with Hind III

Draw a diagram of the gel showing the relative positions of the bands, and label each band with its size in base pairs. This will represent your expected results for the actual digest that you perform.

How much DNA per band?

If you perform a restriction digest and run it on a gel, you'd like to be able to see all the bands. Your ability to see a band on the gel depends on how many nanograms of DNA are in that band, as well as how you stain the DNA. We use ethidium bromide in our gels, which allows us to visualize individual bands containing as little as 50 ng. However, the different bands from a digest won't have the same mass of DNA. If you cut some DNA into two fragments and one fragment is twice as large as the other, the larger fragment will have twice the mass of the smaller fragment.

Suppose you are doing a restriction digest of lambda using the enzyme Hind III. If you start with 500 ng of uncut lambda DNA, how many nanograms of DNA will be in each of the bands on your gel? Label the bands in the Lambda/Hind III lane of your gel diagram with the amount in nanograms. Do you think you'll be able to see the smallest band?

What to turn in

Working with your lab partners, make one pGLO map (showing cut sites for both the unmutated and mutated versions) and one gel picture for your lab group. Label the gel diagram in terms of base pairs and (for Lambda/Hind III only) nanograms. Turn the diagrams in today in lab, with the names of everyone who worked on it. This counts as a quiz.

Review

Terms and concepts

  • Linearize
  • Molecular weight marker. What marker will you use on your plasmid digest gel?
  • Restriction enzyme (endonuclease)
  • Restriction fragments
  • Restriction map. Given a restriction map, you should be able to determine what the gel result would be when DNA is cut with particular restriction enzymes.
  • Restriction site
  • Supercoiled DNA

Review questions

  1. How does a restriction digest help us see the difference between pARO180 and pGLO?
  2. What is the difference between a nucleotide sequence and a DNA map?
  3. On Restriction Analyzer, why does it matter if you check the "linear" or "circular" box?
  4. Suppose you have a linear piece of DNA. You cut it with Hind III, and you get one fragment of 800 bp and one of 200 bp. Draw a restriction map of this DNA.
  5. Is your purified plasmid double-stranded DNA or single-stranded? Relate this to the sequence shown above.
  6. When you are going to perform a restriction digest, how do you determine how much DNA you need to start with in order to see all the bands on your gel? (This is a quantitative question. On a test, I could ask you how much DNA you need to use. What information would you need to answer this question?)
  7. In Bio 6B, you use two different approaches for analyzing your plasmid DNA: the total nucleic acid gel and the restriction digest gel. What are the advantages and disadvantages of each?
  8. Why does it matter if the DNA on a gel is supercoiled?

Resources & References

Bio 6B Flickr site. Find your gel photo and compare it to the photos of other groups.

How to Identify Supercoils, Nicks and Circles in Plasmid Preps BiteSize Bio.

Sequence analysis software

Restriction Analyzer from molbiotools.com. Fairly easy to use.

Restrictionmapper.org. Simple and old-fashioned tool for virtual digests. Clunky, but it works.

Benchling. Outstanding suite of online sequence analysis tools. Requires a free account. This is what I use the most.

Restriction enzymes

Restriction Enzymes from PDB-101. Simple description and molecular diagrams.

DNA Restriction from DNA Learning Center. Brief animated description of restriction, sticky ends, and ligation.

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