"Velcro-DNA" serves as a good model for several reasons:

1. it has two covalently non-identical complementary parts that have "hydrogen bonds" that hold the two pieces together and are easily broken and reformed.

2. Circular "plasmids" can be formed if "sticky ends" are incorporated into the structure you make.

3. "Restriction sites" are easily demonstrated.

4. Genetic "palindromes" can be discussed

5. (Humorously) If you wear a fuzzy sweater, you yourself will make a wonderful demonstration of how single-stranded DNA tightly adsorbs to substrate, such as nitrocellulose binding in the diagnostic blots of various compass directions, and in the mini-DNA chips used in the biotech industry.

Remember that a good model should make all the difficult points conspicuously obvious so that the students don't understand why this is generally thought to be so difficult. Hopefully, this is true here!

Instructor's Set-Up:
Making a v-DNA Plasmid
with one EcoR1 site and two HindIII sites.

1. For purposes of modelling restriction endonuclease activity, a 5-foot (1.5 m) length of WHITE velcro is laid out as shown in Figure 1. The lengths of the two sticky ends are 2 inches (5 cm).

   

2. Since the restriction site for EcoR1 is G'AATTC, with lab marker write the letters AATT at half-inch intervals on the overhanging tab on the left, and then a "C" in the first half-inch of the "double helix". Put a "G" in the last half-inch of the "double helix".

   

3. Then flip over the velcro double ribbon, go to the other side of your table and write AATT on the tab. (Note, the reading directions of the two ribbons are in opposite directions or "antiparallel".) Now add the remaining letters for the EcoR1 site to both ends. (It may help if you join the sticky ends so that you write the letters in the proper directions.) You have done well IF the breaks in the velcro ribbons appears between the G and the A in both instances.

   

4. Next open up the ribbon, and using a long ruler, add the letters for two HindIII sites as shown in Figure 4. Make these sites so that the ribbon is divided into unequal lengths.

   

5. Flip the velcro over and go to the other side of your table to add in the complementary bases of the two HindIII sites.

6. Fill in the remainder of the velcro with a random assortment of bases, lettering always at a constant half-inch interval. (A trick might be useful here so that you don't inadvertantly put in another restriction site: Merely cycle through ATCG over again and again.) Then, and this will take you awhile, write in the complementary bases on the other side of the "double helix."

7. Hook the two sticky ends together to form a belt that has no twists in it. You now ALMOST have a plasmid that has a single EcoR1 site, and two HindIII sites.

8. It is 'almost' a plasmid because there is still one more thing to do: You must convert what you have into a supercoil with two twists in it as shown in this picture borrowed from another place in this site:

   To make the supercoil, carefully open the EcoRi site, rotate one side around twice and stick the site back together again. Now hold the "plasmid" up. Does it hang with two twists in it? It may help to gently shake it a little to allow it to come to equilibrium conformation. Is the supercoil right-handed or left-handed? If it is not like that show in the previous figure, undo the EcoR1 site, get it back to an untwisted belt conformation, and then undo the EcoR1 site and rotate one end twice in the opposite direction you did before. (For some unknown reason, a plasmid is only viable when it has two twists of a supercoil in it.)
   

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A v-DNA Insert Fragment

Obtain a shorter piece of velcro in a contrasting color (don't get too dark a color or the marking pen letters won't show); make "sticky ends" and label them such that they are EcoR1 sites. In the interior of this fragment, add two restriction sites of a third and forth type. Two good blunt-end sites are GG'CC (HaeIII) and for CC'GG (you look up that one's name!). These can easily be added in as CCGGCC on one strand. Then add in a random assortment of other bases by cycling through ATCG again and again until all the spaces are filled. Add the complementary bases to the other side. You now have an EcoR1 restriction fragment. Perhaps it contains the gene for ampicillin resistance (amp^R) so that this model will conform to the common lab exercises used with real plasmids and transformation as employed in teaching.

The Demonstration for Students

1. Obtain a plasmid from the teacher
2. Notice that it is "circular" (is linear but has no end)
3. Hold it up with one hand and allow it to hang loosely. Notice how the circle "wants" to twist around itself a couple of times. That is called a "supercoil." Viable plasmids must have this supercoil built into them. Later we will find out how this supercoiling is done.
4. Moving from the large scale structure to a mid-level scale, you will notice that the "v-DNA" is made of two ribbons of velcro. Each ribbon is non-identical and "complementary" to the other. Two similar ribbons will not stick to each other, but complementary ones will.
5. Notice that the "bases" shown on one ribbon are complementary to those on the other ribbon. A's are opposite to T's; G's opposite to C's.
6. Now let's move down from the mid-range level of structure to the finer genetic code level.
   1. How many sequences of G'AATTC can you find on your plasmid? This is one type of a special sequence called a restriction site. This particular one is called an EcoR1 restriction site because there is an enzyme that will cut this sequence is a particular way,
   2. Now look for two other restriction sites on your plasmid. These are a bit simpler than the EcoR1 site. Unlike EcoR1, which causes sticky ends to be produced, these two are "blunt end" producers. (Note that the apostrophe merely indicates where the restriction endonuclease clips, or "nicks" the polynucleotide chain.)
      1. CC'GG
      2. GG'CC
   3. Palindromes: what are they? Have you seen any genetic palindromes lately? (Yes, all three of the sites so far mentioned are genetic palindromes. Thus your "restrictase" clips both strands. Can you see that?
   4. Ligase is the name of the enzyme that bonds the broken sticky or blunt ends back together again and forms the covalent bonds needed to "heal" the "nicks". (Ligase has the same root as ligature, meaning something that ties things together.)

Questions

In the lab: How can you tell whether or not a plasmid has a particular restriction site, and how many of those sites? Strategy: If the plasmid does not have a single such site, treatment with that enzyme results in absolutely no change to the plasmid. However, if there are one or more such sites, nicks are formed and breakages occur. If there is only one site, does the molecular weight (size) of the plasmid change? How many pieces are formed? If there are two sites, does the molecular weight (size) of the plasmid change? How many pieces are formed? If there are three sites, does the molecular weight (size) of the plasmid change? How many pieces are formed? Back to 'if there were only one site:' is the shape of the plasmid changed? What "molecular biological" methods are available that will sort things by size and shape? After looking through the page called "Analogies", pretend that you applied applicable methods to plasmids not cut, cut only once, and cut three times. You know that you really understand this well if you see that a plasmid cut once acts as if it is of higher molecular weight than if it weren't cut at all! Finally pretend that you treat your plasmid with EcoR1, or with HaeIII (site = GG'CC), or with a mix of both enzymes. How do the products sort out relative to the untreated control according to the sorting method you have chosen. (Once you get through all this, all the hard stuff is over!)

Doing a Bit of Genetic Engineering!

1. Imagine that you have treated the DNA isolated from a frog with EcoR1, and that, of course, resulted in the frog DNA's being cut in many "fragments" of different lengths. How would you sort those into batches of different lengths?
2. What characteristic is common to almost all of the fragments? (Right! EcoR1 sticky ends.)
3. Now suppose that you select one type from all those different fragments (how?), and you mix that particular type of fragment with EcoR1 restricted plasmid, and then add ligase. What do you get? (Show the several possibilities. And from those show which are viable.)

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