Background
Deoxyribonucleic Acid
Less than 50 years ago the nature of the genetic code still eluded scientists. In the 50 years since the structure of DNA was first unraveled, it has become the most significant biological topic of the century. Understanding the structure of DNA helps to explain many life processes and leads to greater knowledge of why we are who we are. In addition, the uniqueness of every individual organism’s DNA can be used as a tool to discover relationships between organisms.
A simplified diagram of a short section of DNA is shown in Figure 1. The diagrammed segment contains seven base pairs. A real chromosome may contain a single DNA molecule with as many as 108 (100 million) base pairs or even more! Since the base pairs represent the genetic code, the chromosomes can store a lot of messages!
{10447_Background_Figure_1_Short DNA sequence}
The two sugar-phosphate backbones in a double standard DNA molecule have opposite orientations. This results because the individual sugar-phosphate backbones have unequal ends. One end is called the 5' (pronounced 5-prime) end, while the other is called the 3′ end. These ends are chemically different. In a double-stranded molecule, one backbone is arranged 5′ to 3′ from top to bottom, and the other is arranged 3′ to 5′ from top to bottom.
Restriction Enzymes
Restriction enzymes, also called restriction endonucleases, recognize and bind to specific base sequences in a DNA molecule and cut the DNA at or near the recognition sequence in a consistent way. The restriction enzymes commonly used in laboratories generally recognize specific DNA sequences of 4 or 6 base pairs. These recognition sites are palindromic in that the 5′-to-3′ base sequence on each of the two strands is the same. Most of the enzymes make a cut in the backbone of DNA at a specific position within the recognition site, resulting in a break in the DNA. These recognition–cleavage sites are called restriction sites.
Figure 2 shows some examples of restriction enzymes and their recognition sequences. The arrows indicate the cut sites and the names indicate the organism from which the enzymes were purified (for example, EcoRI from Escherichia coli).
{10447_Background_Figure_2_Restriction enzyme examples}
Notice that the “top” and “bottom” strands read the same from 5′ to 3′; this characteristic defines a DNA palindrome. Also notice that some of the enzymes introduce two staggered cuts in the DNA, while others cut each strand at the same place. Enzymes like SmaI that cut both strands at the same place are said to produce blunt ends. Enzymes like EcoRI that leave DNA fragments with single-stranded protrusions are said to produce sticky ends.
5′ G AATTC 3′
3′ CTTAA G 5′
DNA Ligase
If a DNA molecule has been “digested” by different restriction enzymes into fragments, the ends of the DNA fragments will clearly not all be the same. If, per chance, the ends of the fragments from different DNA molecules do match perfectly, then the question is: Can they be reunited into a new recombinant DNA molecule? Cells contain an enzyme that can expedite this reuniting process. The enzyme is called DNA ligase. If two pieces of DNA line up perfectly, DNA ligase can help form the bonds between the sugars and phosphates to seal up the chemical backbone of the molecule and create a new DNA molecule.
Recombinant DNA
Some of the most important techniques used in biotechnology laboratories today involves making recombinant DNA molecules. Recombinant DNA molecules are DNA structures that have been reassembled from DNA fragments taken from more than one original source. Restriction enzymes and DNA ligase are important tools in executing the recombinant work. Look at the two molecules below (R and S) to visualize how a BamHI restriction enzyme might help to produce a new, recombinant R/S DNA molecule. Note that the Rs and Ss represent base pairs in R and S DNA molecules.
{10447_Background_Figure_3}
When biologists make recombinant DNA molecules, they usually purify the starting DNA molecules and work with them in test tubes. The starting DNA molecules are first digested using restriction enzymes. The resulting pieces and DNA ligase are then mixed together. The ligase forms bonds between fragments with complementary ends.
Transformation
Biologists try to introduce new DNA into cells by trying to get the cells to absorb the DNA that they have placed in their surrounding environment. If a cell absorbs the “foreign” DNA and incorporates it into its genome, it is said to be transformed. Cell types vary considerably in their ability to absorb DNA from their environment. Some bacterial cells are relatively adept at transformation and have become the favorite vehicle for geneticists.
It is possible to introduce plasmids (circular DNA molecules with an origin of replication) into bacterial cells through the process of transformation. Bacteria that can be transformed (can take up new DNA) are called competent. Some bacteria are naturally competent and others can be made competent by chemical and physical treatments. Once the bacteria absorbs the plasmid DNA, they copy it along with their normal DNA when they reproduce. The net result is that the geneticist has a continuing source of the new DNA (by recovering it from the transformed cells). Because many identical copies of the new DNA are generated in this process it is often called cloning.
If only this process were as easy as it sounds. Most cells will not maintain and copy just any DNA molecule. The DNA molecule must meet specific conditions of the host cell. In the case of E. coli, a very commonly used host, the DNA must be a circular molecule like that in the bacteria and it must have an origin of replication. An origin of replication is a special sequence of bases where the copying (replication) begins.
A typical experiment used for proof of transformation involves adding a plasmid with a gene for antibiotic resistance to serve as a “marker” in a newly transformed bacteria. A bacteria that cannot normally grow on a media containing an antibiotic is transformed into a bacteria that can grow on a media with the antibiotic. The actual growth on the media is considered to be proof that the bacteria has been transformed. Using this “marker” is very effective since the transformed bacteria are the only ones that can grow on the media containing the antibiotic—bacteria that have not been transformed cannot grow! The other reason this technique is so significant is that transformation is usually very inefficient. In a typical experiment, less than one cell in 1,000 will be transformed, but once transformed, it can multiply and produce many more bacteria with the desired gene.
Materials
Plasmid Simulation Worksheet
Pop beads, blue, 58
Pop beads, green, 10
Pop beads, orange, 12
Pop beads, pink, 8
Pop beads, red, 8
Pop beads, yellow, 8
Twist ties, 8–10
Procedure
Modeling Plasmids
Your team will model one ampicillin-resistant plasmid (pAMP) and one kanamycin-resistant plasmid (pKAN). Each plasmid will have two identically-colored DNA strands. Be sure that one strand is put together 5′ to 3′ while the other is 3′ to 5′. The orientation will be modeled with the hole (3′) and the protrusion (5′) end on the pop beads. Before the plasmids are formed into circles, the two strands should be lined up perfectly and tied together with twist ties as shown in Figure 3. Wrap the beads with twist ties about every three to four beads but do not wrap them between the two red or the two yellow beads. These two colors are reserved for the restriction (separation) sites.
{10447_Procedure_Figure_3_Plasmid model—double stranded circular molecules}
Examine the sequences shown in Figure 4 for the order of the pop beads in the plasmid models. Each pop bead represents approximately 200 bases of DNA, except for the restriction sites. Each restriction site is represented by a pair of uniquely colored beads (yellow for HindIII and red for BamHI). Once the parent plasmids are constructed (completed double-stranded circular plasmid) go to the Digesting Plasmid section. Orange beads designate the origin of replication site of each plasmid. Green beads represent the KAN-resistant gene and pink beads AMP-resistant gene.
{10447_Procedure_Figure_4_Model plasmid sequence}
Digesting Plasmids
- Digest each plasmid with HindIII. Simulate this by popping apart the two yellow bead pairs representing the HindIII recognition sites. Each plasmid should give a linear chain of beads with yellow beads on either end.
- Next digest each plasmid with BamHI by separating the chain in between the two red beads in each chain. This should result in a total of four linear DNA fragments. Each fragment should have a pair of red beads at one end and a pair of yellow beads on the other end. There should be a 5′ (protrusion) and a 3′ (hole) end at each end of the fragments.
- Now pool the DNA fragments with one or more other work teams as directed by your instructor. Place the fragments on a table top (this will simulate a test tube full of digested fragments).
Recombination Patterns
- Now simulate the activity of DNA ligase. Remember DNA ligase will only reformulate complementary base pairs like those originally split by HindIII and BamHI. Further, it is important to note that once two HindIII ends have been rejoined that the new DNA molecule will have two free BamHI ends. It is more likely that these free BamHI ends on the same molecule will join each other to form a new circular plasmid than it is for an additional fragment to join in a linear fashion. In other words, it is unlikely that any product DNA will be formed from more than the four starting pieces. In fact, most new DNA molecules will result from random selections of two fragments.
- Focus on all the different circular products that can be made by joining any two of the pooled fragments together. Hint: There are 10 products possible. Use the information on top of the Plasmid Simulation Worksheet and draw all ten possible products and fill in the answers required on the worksheet.
