Biology 2960 Computer Laboratory

Restriction Digest Map



"THE BIG PICTURE"

Suppose we have a circular DNA molecule, called pTut (for "tutorial"), that is 7666 bp (7.666 kbp) long. (1 kbp = 1 kilobase pair = 1000 base pairs.) We are going to prepare a restriction map for the sites for the restriction enzymes BglII, BstEII, and EcoRV. This map will show the locations of the cutting sites of the three enzymes with respect to one another.



BACKGROUND

Plasmids

Plasmids are small, circular DNA molecules which are physically separate from chromosomes. They are often found in simple organisms like E. coli, algae, and yeast. They can vary in size from a few thousand bp to several hundred thousand (bp) base pairs. Plasmids replicate using their own replication origins and gene products (proteins and RNAs) and can transfer themselves to other bacterial cells through the process of conjugation (bacterial mating). They often carry genes that encode resistance to one or more antibiotics and can confer this drug resistance to their bacterial hosts, making plasmids clinically important. Molecular biologists have used plasmids to serve as vectors which carry "foreign" DNA fragments spliced or inserted ("cloned") into the plasmid. (NOTE: you will be performing this cloning experiment near the end of the semester). Lastly, because plasmids are very small in size compared to bacterial and yeast chromosomes, they can be easily isolated separately from chromosomes using special procedures.


Restriction Enzymes

Restriction enzymes, also called restriction endonucleases, recognize specific base sequences in double-helical DNA and cleave, at specific places, both strands containing the recognized sequences. Many restriction enzymes recognize specific sequences of 4 to 8 base pairs and hydrolyze a phosphodiester bond in each strand in the region. A striking characteristic of these "cleavage sites" is that the recognition sequence is palindromic. A palindromic sequence is a sequence which is the same when read on both strands in the same (5'-to-3') direction. In addition, the actual phosphodiester bonds broken are symmetrically positioned within the sequence (see below).


The arrows are the points at which the enzyme breaks the phosphodiester bond in the backbone of the DNA. Once the backbone is broken, the hydrogen bonds are not sufficient to hold the strands together, and they separate.

One powerful technique in molecular biology is physically mapping DNA molecules with restriction endonucleases. In 1979, Nathans, Smith and Arber were awarded the Nobel Prize for discovering restriction enzymes and having the insight and creativity to use these enzymes to map genes. In today's lab, you will construct a very basic restriction map of the plasmid pUC19, which is a small (2686 bp) vector derived from a naturally-occurring E. coli plasmid.



PROCEDURE

Step 1: Cutting the DNA


Cut the DNA with each enzyme singly, and with all possible pairwise combinations.

Lane
1. pTut x BglII
2. pTut x BstEII
3. pTut x BglII + BstEII
4. pTut x EcoRV
5. pTut x BglII + EcoRV
6. pTut x BstEII + EcoRV
7. Lambda x HindIII: 23,130, 9416, 6557, 4361, 2322, 2027, and 564 bp fragments.

(The 125 bp fragment usually runs off the gel or is too faint to be seen.)

Step 2: Gel Electrophoresis - Analyze samples of the restriction digests, along with a marker, by agarose gel electrophoresis.

Step 3: Visualizing the Bands - Using ethidium bromide and UV light exposure, visualize the DNA bands and take a photograph. (Note: ethidium bromide was incorporated into your agarose gel in lab.)

Step 4: Determining the Lengths of the DNA Fragments - Determine the DNA fragment sizes. If necessary, construct a calibration curve for the marker data, measure the migration distances for bands in the experimental lanes, and use the calibration curve to determine the DNA fragment sizes. Enter the results in a Table:
Digestion Results
1 2 3 4 5 6
BglII BstEII BglII + BstEII EcoRV BglII + EcoRV BstEII + EcoRV
7666 7666 6008 4729  4729 4016
1658 2937 1992 2937
945 713



ANALYSIS

Step 1: BglII (lane 1) and BstEII (lane 2) fragments - The result in each case is a single frament of 7666 bp. Therefore we can draw very simple restriction maps for these two enzymes: (Note: This does not mean the sites are in the same place, as we will see.)
Step 2: BglII and BstEII together (lane 3) fragments (6008 and 1658 bp) - Conceptually, you can visualize how we made this map by considering two disks, one with the BglII site, and the other with the BstEII site that we overlaid and rotated (clockwise) until BglII and BstEII were 1658 bp apart. Thus the two restriction sites can be placed on a map as shown in the figure to the right. (Note: the map could also be drawn as a mirror image if we were to rotate the BstEII disk counter-clockwise.)

REPLAY ANIMATION


Step 3: EcoRV (lane 4) fragments (4729 and 2937 bp) - The EcoRV map, again without putting the sites at absolute locations.

Step 4: EcoRV and BglII together (lane 5) fragments (4279, 1992, and 945 bp) - Comparing this with EcoRV cutting alone, we can conclude that the BglII site must be in the 2937 bp EcoRV fragment, cutting it into the 1992 and 945 bp pieces (1992+945=2937). Using our rotating disks method, we can create an EcoRV and BglII map. (Note that an alternate map with the BglII site 945 bp away, clockwise, from the "top" (12 o'clock) EcoRV site is also possible.)

REPLAY ANIMATION

Step 5: EcoRV and BstEII together (lane 6) fragments (4016, 2937, and 713 bp) - Comparing this with EcoRV cutting alone, we can conclude that the BstEII site must be in the 4729 bp EcoRV fragment, cutting it into the 4016 and 713 bp fragments (4016+713=4729). Using our rotating disk method, we can create an EcoRV and BstEII map. ( Note that an alternate map with the BstEII site 713 bp away, clockwise, from the "bottom" (5 o'clock) EcoRV site is also possible.)

REPLAY ANIMATION


Step 6: Constructing the final map (part 1): Combining the EcoRV+BglII map and EcoRV+BstEII map - If we simply overlay the EcoRV+BglII map (step 4) with the EcoRV+BstEII map (step 5), the calculated fragment sizes for a BglII+BstEII digest (lane 3) would be 4016 and 2705 (map on the left). However, recall from the BglII+BstEII cut data, we see that the actual fragments produced are 6008 and 1658 bp (see Step 2 above and map on the right). Therefore, we need to use one of the alternate maps as mentioned in Step 4 and Step 5.

REPLAY ANIMATION


Step 7: Constructing the final map (part 2): Combining the EcoRV+BglII map and EcoRV+BstEII map - Combining the alternate map from Step 5 in which the BstEII site 713 bp away, clockwise, from the "bottom" (5 o'clock) EcoRV site with the EcoRV+BglII, we can observer that the calculated fragment sizes for a BglII+BstEII digest (lane 3) would be 6008 and 1658, which matches the BglII+BstEII cut data (lane 2).

We have now completed our restriction map.
HURRAY!!! Go Bio... go Bio... it's your birthday... go Bio!
REPLAY ANIMATION


Answer Question #9 and #10 in your lab report. For Question 10, use the gel image below as your data.

Adapted from Pearson Education, Inc. publishing as Pearson Prentice Hall © 2007 All Rights Reserved