GENETICS LAB III

In the last lab you isolated plasmid DNA from E. coli. You were told that plasmids and chromosomes were different, but what does it mean to be a plasmid? What does it mean to be a chromosome? The analysis you will do in this lab may provide some insights into these questions.

A. Restriction mapping: A restriction map is a description of restriction endonuclease cleavage sites within a piece of DNA. Generating such a map is usually the first step in characterizing an unknown DNA, and a prerequisite to manipulating it for other purposes. Typically, restriction enzymes that cleave DNA infrequently (e.g. those with 6 bp recognition sites) and are relatively inexpensive are used to produce a map.

The DNA to be restriction mapped is usually contained within a well-characterized plasmid or bacteriophage vector for which the sequence is known. In fact, there are usually multiple known restriction sites immediately flanking the uncharacterized DNA, which facilitates making the map. In this lab, unknown DNA will be placed into a well-known plasmid. We want to get an idea of what the unknown DNA looks like (i.e., it’s sequence). So, the plasmid will be digested with various restriction enzymes and then the digested DNA will be separated on a gel. By looking at the size of the bands produced, you will be able to get an idea of the unknown DNA’s sequence. Here is an example:

“To illustrate these ideas, consider a plasmid that contains a 3000 base pair (bp) fragment of unknown DNA. Within the vector, immediately flanking the unknown DNA are unique recognition sites for the enzymes KpnI and BamHI. As illustrated in the figure below, consider first separate digestions with KpnI I and BamHI I:

Digestion with KpnI I yields two fragments: 1000 bp and "big". Since there is a single KpnI I site in the vector, the presence of a 1000 bp fragment tells you that there is also a single KpnI I site in the unknown DNA and that it is 1000 bp from the KpnI I in the vector. The "big" fragment consist of the vector plus the remaining 2000 bp of the unknown.

Digestion with BamHI I yields 3 fragments: 600, 2200 and "big". The "big" fragment is again the vector plus a little bit (200 bp in this case) of unknown DNA. The presence of 600 and 2200 bp fragments indicate that there are two BamHI I sites in the unknown. You can deduce immediately that one BamHI I site is 2800 bp (600 + 2200) from the BamHI I in the vector. The second BamHI I site can be in one of two positions: 600 or 2200 bp from the BamHI I site in the vector. At this point, there is no way to know which of these alternative positions is correct.

The trick to determining where the second BamHI I site is located is to digest the plasmid with KpnI I and BamHI I together (click the diagram below with your mouse to see this effect). This so-called double digest yields fragments of 600, 1000 and 1200 bp (plus the "big" fragment). The 600 bp fragment is the same as obtained by digestion with BamHI I alone. The 1000 and 1200 bp fragments tell you that KpnI I cut within the 2200 bp BamHI I fragment observed when the plasmid was cut with BamHI I alone. You already know where KpnI I cuts in the unknown DNA, and you therefore now know the location of the second BamHI I site!

If the process outlined above were conducted with a larger let of enzymes, a much more complete map would result. In essence, single digests are used to determine which fragments are in the unknown DNA, and double digests to order and orient the fragments correctly.

Success in using this technique depends upon obtaining complete digestion of the DNA with each of the enzymes used! Partial digestion will yield fragments that are ultimately a great source of confusion. One way to avoid this problem is to add up the estimated sizes of all the fragments in each lane - if they don't sum to roughly that of the intact DNA, it is likely that digestion was not complete. One other thing to watch for is the presence of two fragments of roughly the same size that may appear to be one fragment on an agarose gel. This situation is often suspected by observing an abnormally bright fragment on an ethidium-stained gel, or by a fragment being broader than expected.” (http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/maps.html)

Restriction enzymes have been the most important tool in the incredible technology of genetic engineering that has emerged in the last 20 years. It is difficult to appreciate how they work. If you get an intuitive feel for how restriction enzymes work on DNA, you have gone a long way to gaining a sense of what is possible in genetic engineering. Most restriction enzymes used in the laboratory recognize short palindromic DNA sequences and cut the DNA somewhere within the recognized sequence. It is possible to examine a DNA sequence and predict with near certainty whether a given restriction enzyme will cut. For example, HaeIII, with a recognition sequence of ATGGCCGTT will almost certainly cut a DNA sequence ATGGCCGTTTACCGGCAA.

Restriction enzymes all require magnesium for activity, but they differ in what pHs and salt concentrations they prefer. Therefore, it is important to use the buffer that corresponds with a particular restriction enzyme. EDTA sequesters magnesium, making it unavailable to restriction enzymes, hence stopping the cutting reaction.

In this lab, we will be using restriction enzymes to digest (cut) our plasmid DNA, yielding a precise number of DNA fragments. Keep in mind that cutting a circular piece of DNA (e.g. a circular plasmid) gives one fragment; cutting a circle twice gives two fragments, etc. We will then use electrophoresis to view the results of our restriction enzyme digestion.

Enzyme	Recognition Sequence	# of Sites found	Cleavage sites
HindIII	AAGCTT	1	In the lacZa

B. Visualizing your fragments:

Electrophoresis permits you to see something directly related to the physical and informational properties of DNA. In electrophoresis, a voltage is applied across a gel. Negatively charged molecules (like DNA) migrate towards the positive electrode. DNA diffuses very slowly in an agarose gel, so it hardly moves except from the force of the electric field. The larger a piece of DNA is, the more slowly it will move through the gel. Conversely, small pieces of DNA will migrate through the gel more rapidly. The relationship between the size of a DNA fragment and the velocity it travels through a gel is complex, but it is close to logarithmic. The log of the size of the fragment is more or less inversely proportional to the velocity of the fragment and hence to the distance traveled at the end point.

DNA can't be seen directly, but ethidium bromide, a dye that fluoresces orange when UV light shines on it, binds to DNA and so permits you to visualize the location (and relative abundance) of DNA. The more DNA at a location, the more intense the fluorescence. Xylene cyanol and bromophenol blue (found in the Stop solution) are, like DNA, negatively charged and so migrate to the positive electrode. Unlike DNA, they are colored, so you can use them to monitor the progress of the electrophoresis. Bromophenol blue migrates at about the position of a DNA fragment several hundred base pairs in length.

3. Hold pipetter in a vertical position, with the tip facing down. Measure accurately.

B. pUC19 map: pUC19 is a commonly used E. coli plasmid cloning vector. The molecule is double-stranded circle of 2686 base pairs in length and carries a multiple cloning site that contains unique sites for 13 different restriction endonucleases (6-cutter).

1. Digestion of Nucleic Acid

1.1 Prepare digestion: Each group from last week will make two digestion reactions. One digestion reaction will use HindIII to cut pUCP18. The digestion will be made in a final volume (=reaction volume) of 20ul. A general digestion reaction is composed of the following :

1.2 Incubate: Let digestion incubate in a water bath at 37°C for 2-3 hrs. (At this point, do step 2.1.)

1.3 Stop reaction: Remove tubes from water bath and to each tube add 3µl of Stop solution. Mix tubes and spin down liquid briefly (5 sec).

2.1 Pour a 1% Agarose gel: Assemble electrophoresis apparatus. Combine 75ml TAE and .75g Agarose, heat in microwave (careful it doesn’t boil over). When hot and the agarose is completely melted, add 3µl of Ethidium bromide or Sybr Safe, and pour into mold. Let stand for 1 hour.

separate well in the gel. In order to do this, place the tip of the pipette just inside the well. If you stick the tip of the pipette all the way to the bottom of the well, you will puncture the well and your DNA will leak out. Record the positions of your samples.

2.4 Begin Electrophoresis: turn on voltage to 120 volts. Let it go for about 45min (monitor the position of the blue dye). If the voltage is less (say 95 volts), then it will take longer for the DNA to travel.

2.5 Take a picture of the gel. Your TA will take you to a darkroom with a transilluminator so that you can see your gel in living color and take a picture of it. You will need to be able to interpret the results.

How could you isolate the entire gene on a small fragment? How big would the fragment be?