Biology 115 (Survey of Biology Laboratory) Spring 2006
Lab 10: Gene Technology
DNA
DNA is the hereditary molecule. The information necessary to build an organism is encoded in the bases of a DNA molecule. Deoxyribonucleic Acid (DNA) is composed of subunits called nucleotides. A single nucleotide has three parts: a phosphate group, a 5′-carbon sugar (deoxyribose) and a nitrogenous base. There are four different nitrogenous bases in DNA: adenine, thymine, guanine, and cytosine. These four bases can be placed into two groups based on the structure of the base. Two of these, adenine and guanine, are in a double-ring structure. These two bases are classified as purines. The other two bases, cytosine and thymine, are single-ring bases and are classified as pyrimidines. Nucleotides (sugar, phosphate and base) are linked together into long chains by covalently bonding the phosphate of one nucleotide to the sugar of the next nucleotide. This results in a sugar-phosphate “backbone” with the nitrogenous bases hanging off to the side.
DNA is a double-stranded molecule. Thus, there are two of these sets of linked nucleotides, with the sugar-phosphate backbones running up and down on the outside, and the nitrogenous bases pointing off toward the middle of the molecule. The nitrogenous bases form hydrogen bonds with each other across the middle, and form something like the “steps” of a ladder. On this “ladder”, the sides of the ladder would be the sugar-phosphate backbones, and the steps would be made from one nitrogenous base on one side hydrogen bonded to the nitrogenous base from the other side. The nitrogenous bases will only form hydrogen bonds with certain other bases. In fact, adenine will only form hydrogen bonds with thymine, and cytosine will only form bonds with guanine. Thus, the base-pairing rules for DNA are A with T and C with G. Notice that, because A and G are double-ring bases (purines), and C and T are single-ring bases (pyrimidines), the base-pairing rules mean that a purine always forms hydrogen bonds across with a pyrimidine. In other words, a double-ring always bonds with a single-ring base. This has the effect of keeping the sides of the “ladder” (i.e. the sugar-phosphate backbones) exactly the same distance apart. Additionally, because the base-pairing rules are completely specific, it means that only one side of a DNA molecule is needed to completely re-create the other side. In fact, this is how DNA is copied when cells divide: the two sides are split apart (i.e. the hydrogen bonds between the bases in the middle of the “steps” of the ladder break) and each side is used as a “template” to build a new molecule.
The information in a DNA molecule is encoded in the sequence of the nitrogenous bases. Each organism has a slightly different sequence of bases from every other organism (except for genetically identical clones). Even among clones, random errors in DNA replication (mutations) can result in slight differences in DNA sequence. Differences in the sequence of bases can be identified by separating fragments of DNA in a process called gel electrophoresis. This is the basis of genetic fingerprinting. In today’s lab, you will perform gel electrophoresis and identify fragments of DNA that are different sizes and thus, have different sequences of nitrogenous bases.
Gel Electrophoresis
The principle behind gel electrophoresis is fairly simple. DNA is a negatively-charged molecule and if fragments of DNA are placed into an electrical field, the DNA will be attracted to the positive end of the field. If you put the DNA into a medium (say, a 0.8% agarose gel – a jello-like substance) and then turn on the electric field, the DNA fragments will move toward the positive end, but will have to maneuver in-between the molecules of the gel. Smaller DNA fragments will more easily be able to maneuver between the gel molecules, and so will move more quickly toward the positive end of the field. Larger fragments will move more slowly. This means that, if we apply the electrical field for a fixed amount of time, the DNA fragments will separate out along the gel by size. Smaller fragments will have moved farther along the gel than larger fragments. Then, if we can stain the fragments of DNA somehow, we can see bands of DNA on the gel that correspond to fragments of different length. This is exactly what we’ll do today.
Plasmids
The DNA that we’ll be using comes from a plasmid. Plasmids are small, circular DNA molecules that occur in bacteria, and are replicated independently of the bacterial chromosome. We can get large amounts of plasmid DNA by growing bacteria containing the plasmid in culture, and then purifying the plasmids from the culture. All of the DNA we’ll use today is from multiple, identical copies of a plasmid obtained this way.
Restriction Endonucleases (Restriction enzymes)
Restriction endonucleases are enzymes that cut DNA at specific sequences. Each endonuclease recognizes a unique sequence of bases, and cuts both strands of DNA (both sides of the ladder) at that sequence. For example, one endonuclease might cut DNA every time it sees the sequence GAATTC, while another might cut every time it sees ACTAGT. The fact that each endonuclease cuts at a specific sequence means that every time identical DNA is cut by one endonuclease, the cuts are at exactly the same place and the fragments of DNA produced are exactly the same size.
DNA Fingerprinting
Molecular biology can be used to identify particular individuals. In the DNA fingerprinting technique, a specific region of DNA is analyzed using restriction fragment length polymorphism (RFLP). This technique makes use of polymorphisms (naturally occurring minor differences in DNA sequences that occur within or between the restriction endonuclease recognition sites). Differences in DNA sequence that result in different restriction fragment patterns are scattered throughout genomes, including that of humans. These are known as RFLPs. Restriction fragment length polymorphisms are made use of in criminal cases, paternity suits, and in diagnosing genetic disorders. The DNA is first cut with a restriction endonuclease, the fragments are separated by gel electrophoresis, the double helix of DNA is then separated to make it single-stranded, and a radioactive probe (also single-stranded) that has a sequence that is complementary to the DNA of interest is then hybridized (bound) to the DNA. The probe will only bind to the DNA that is complementary in sequence. The unbound probe is washed off. Photographic film is then laid over the gel and the radioactivity of the bound probe exposes the film, forming an image corresponding to the fragments of interest (where the probe has bound). The restriction pattern is then analyzed. We will not use radioactive hybridization in this lab. Instead, the fragmentation patterns will serve as fingerprints, and the fragments of DNA will either be stained by methylene blue or by ethidium bromide, a fluorescent dye that binds DNA.
Procedure
The gels are 0.8% agarose and have already been poured and are ready to use. Additionally, the sample DNA has already been incubated with the restriction endonucleases. DNA has thus been cut with restriction enzymes and the fragmentation patterns serve as individual fingerprints. A tracking dye has been added to each of the samples so that we can watch the progress of the DNA as it moves along the gel. We cannot actually see the DNA, but we can see the dye molecules separate along the gel and we will turn off the electric field when the farthest dye band gets near the end of the gel (we don’t want to migrate all of our DNA off the end of the gel).
Each group of students will be responsible for loading, running, and staining one gel. Each student will first practice loading a well using practice gels. The group will then load their “real” gels, with each student loading AT LEAST one well. There are two “mysteries” to solve. Half the lab groups will run the gels for the crime scene DNA and the other half will load the gels for the paternity testing DNA. At least one gel for each mystery must be stained with ethidium bromide. The remainder will be stained with methylene blue. Every student will be responsible for the data in both gels!
The samples are as follows:
Crime Scene
Lane / Sample / Description1 / A / DNA from crime scene 1 cut with enzyme 1
2 / B / DNA from crime scene 1 cut with enzyme 2
3 / C / DNA from suspect 1 cut with enzyme 1
4 / D / DNA from suspect 1 cut with enzyme 2
5 / E / DNA from suspect 2 cut with enzyme 1
6 / F / DNA from suspect 2 cut with enzyme 2
Paternity Testing
Lane / Sample / Description1 / A / Standard DNA fragments
2 / B / Mother cut with enzyme
3 / C / Child cut with enzyme
4 / D / Father 1 DNA cut with enzyme
5 / E / Father 2 DNA cut with enzyme
The gels are sitting in plastic trays and are covered with a fluid. The fluid is a TBE buffer that has free ions in it so that the electrical field can be conducted through the gel. Each gel has eight small wells at the top. These are small depressions in the gel and it is into these wells that you will load your DNA sample. The tracking dye is heavier than the TBE buffer, so when you inject your DNA sample, it will drop through the buffer down into the well in the gel. When we turn on the electric field, the DNA fragments and the dye molecules will begin to move through the gel matrix.
Loading the gel
Students will use micropipettes to load exactly 20μl (20 micro-liters) of DNA sample into each well. Your instructor will show you how to use the micropipettes, but be CAREFUL with them! These are expensive, precision instruments, and dropping one is all that it would take to ruin it. The tips for these micropipettes are disposable, and generally only a single tip would be used for each loading, but we will re-use the tips and will clean them between loadings by pumping the micropipette a couple of times in the buffer solution. Be careful NOT to puncture the gel! You should load into the well, not push the pipette tip through the agarose.
After the gels are loaded, we will put the tops on the electrophoretic apparatus and turn on the current. It will take AT LEAST 30 minutes for the DNA to separate out along the gel. We can watch the progress by noting the bands of dye as they move along. When the dye has moved almost to the end of the gel, we’ll turn off the current.
Staining and viewing the gel
Now we need to stain our DNA fragments so that we can see them. One way to stain the gels is to soak them in methylene blue (a dye that stains DNA) and then look for the blue bands under normal light. We will do this for most of our gels. However, we will also stain at least two of our gels with a chemical called Ethidium Bromide. Ethidium bromide also binds to DNA and it fluoresces under ultraviolet light (however, it is invisible under normal light). Ethidium bromide is also a mutagen, which means that it damages DNA (BE CAREFUL-it will damage your DNA if it comes into contact with your cells!!!), so your instructor will stain the gels, wearing gloves.
Methylene Blue Technique
1. Remove the gel from its bed and place on part of the bench that is covered with plastic wrap. Wet the surface of the gel with buffer.
2. Wearing gloves, place the blue dye side of the Instastain® Methylene Blue card on the gel.
3. Firmly run your fingers several times over the entire surface of the card to establish good contact between it and the gel.
4. Place a gel casting tray and weight on top of the card. This will ensure continuous contact between the card and the gel.
5. Stain for 5-10 minutes, then remove the card. If the gel appears to be only very lightly stained, wet the surface with buffer and replace the card for an additional 5 minutes.
6. Transfer the gel to a dish of distilled water (covering the gel). The water can be changed as necessary to complete destaining.
7. Examine the gel on a light box.
Ethidium Bromide Technique
1. Remove the gel from its bed and place on part of the bench that is covered with plastic wrap. Moisten the gel with a few drops of electrophoresis buffer.
2. Wearing gloves, place the unprinted side of the Instastain® Ethidium Bromide card on the gel.
3. Firmly run your fingers several times over the entire surface of the card to establish good contact between it and the gel.
4. Place a gel casting tray and weight on top of the card. This will ensure continuous contact between the card and the gel.
5. Stain for 10-15 minutes, then remove the card. If the gel appears to be only very lightly stained, wet the surface with buffer and replace the card for an additional 5 minutes.
6. Transfer the gel to a short wave (300 nm) ultraviolet illuminator for viewing DNA. Close the lid (uv is damaging to the eyes)! Turn off the light to view. If the gel has not been sufficiently stained the staining procedure can be repeated.
7. Photograph in the Butler lab.
Results
Using the gels determine;
a) Which suspect committed the crime
b) Which male is the father of the child
Turn in your answers, with an explanation for your choice.
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