We have to use DNA technology to clone the gene we want to study. It's a necessary evil. After the gene is cloned, we can get to the good stuff and look at different gene sequences and expression. We can use the following tools and techniques to get to the bottom of gene expression.
Restriction fragment length polymorphism (RFLP; pronounced "rif lip") is used to distinguish the difference in sequence between alleles for a gene. Sometimes these differences are located within a sequence recognized by a restriction enzyme. If this is the case, we can cut the DNA with a restriction enzyme and then do DNA electrophoresis. Different patterns of DNA pieces on the gel would indicate a rif lip.
A Southern blot (named for the scientist Edwin Southern) can be performed to look for the presence of a specific DNA sequence. In this technique, we treat different DNA samples with restriction enzymes and then use gel electrophoresis to separate the fragments. The DNA is transferred from the gel to a piece of paper-like material called nitrocellulose. This is the blot part.
We make a short sequence of DNA. This sequence is called a probe and is labeled or tagged in some way so it can be detected. It is incubated in solution with the nitrocellulose. The single-stranded probe binds its mate, or its complementary DNA. Its tag or label can then be detected.
Image from here.
What if we want to know if a particular gene is being transcribed into mRNA? One of the experiments that we can do is a relative of the Southern blot, called the Northern blot. Clever, right? The principle of the Northern blot is similar to the Southern blot.
In a Northern blot, we perform electrophoresis of mRNA. We then transfer the mRNA on the gel to nitrocellulose and use an mRNA probe to find its mate.
While Northern blots are still useful, there is another more sensitive and quantitative technique to detect mRNA, called reverse transcriptase-polymerase chain reaction (RT-PCR).
We're getting a little ahead of ourselves. Before we can discuss RT-PCR, we have to talk about P to the C to the R. Polymerase chain reaction (PCR) is used to amplify a DNA sequence. In fact, it can make billions of copies in just a few hours!
PCR consists of three steps per cycle:
- Denaturation: Temperature is raised. Double stranded DNA doesn't like this, and the two strands separate.
- Annealing: Short sequences complementary to the DNA sequences on opposite ends of a gene, or primers, bind to the DNA.
- Extension: Enter the king of all DNA polymerases, Taq polymerase. Taq adds complementary bases to the 3' end of the primers.
Image from here.
The number of molecules formed is equal to 2n where n is equal to the number of cycles. The bottom line is this: you want amplification of your piece of DNA? PCR is your man.
Now we can talk about RT-PCR. This technique serves the same purpose as a Southern blot, but requires smaller amounts of starting material. The mRNA is incubated with an enzyme called reverse transcriptase (RT). RT creates complementary DNA (cDNA) from mRNA. The rest is the same as normal PCR. The DNA is amplified and fragments can be separated by electrophoresis on a gel.
Yet another method to detect mRNAs is in situ hybridization. In situ hybridization recognizes the location of a particular mRNA. We can make a labeled (usually fluorescent) probe complementary to an mRNA that will recognize and bind to this sequence wherever it is located. One of the great features of this technique is the colorful data that we get out of it. It gets pretty boring looking at bands on a gel. Bring on the color!
Image from here.
We've discussed Southern and ever so aptly named Northern blots. What about Western and Eastern blots? Well, if we want to go a step further than RT-PCR or in situ hybridization and look for the presence of a particular protein, we can do a Western blot, which is also called an immunoblot.
We take a sample extract from cells and incubate it in a buffer that causes the proteins to unfold (or "denature"). We then separate the samples by polyacrylamide gel electrophoresis that also involves sodium dodecyl sulfate, or SDS. SDS helps to further denature the proteins in the sample so they can be separated by size.
After the gel is finished running, the proteins are transferred to nitrocellulose. Sound familiar? After the transfer, we incubate the blot with an antibody that is specific only for the protein of interest. Assume that the protein of interest is "actin." Actin would be called the primary antibody.
The blot is then rinsed and incubated with a secondary antibody against the animal that produced the first antibody. Confused yet? If our primary antibody for actin was made in rabbit, we would use a secondary antibody made in another organism against rabbit antigens. This antibody is able to recognize the primary antibody and specifically bind to it.
Why bother using a second antibody? The secondary antibody is bound covalently to the protein alkaline phosphatase which eventually will help us to "see" the protein.
Now the primary antibody is bound to the protein it recognizes on the blot, and the secondary antibody is bound to the primary antibody. It sounds like the start of a conga line, doesn't it? Alkaline phosphatase is attached to the secondary antibody and is chomping at the bit to catalyze a reaction. It's an enzyme, after all.
A substrate is added that forms a solid or precipitate after alkaline phosphatase is finished with it. The precipitate cannot be seen with the naked eye, but it can be picked up by a specialized instrument. The instrument confirms the presence (or absence) of the protein.
Image from here.
One of the newest and perhaps most powerful techniques that can be used to analyze gene expression is the superman of all techniques, the DNA microarray. Often called gene chips or DNA chips, these microarrays contain representative sequences of several thousand genes.
We can collect mRNA from cells and use it to make fluorescently tagged cDNA. The cDNA is then applied to the microarray. Base pairing between the DNA probe sequence on the array and the cDNA would be a match. We use a special machine to scan the chip. The brightness of the fluorescent spot where there is a match correlates with the level of expression.
Aside from the fact that it's just really cool to screen thousands of genes in a single experiment, why would we want to perform microarray analysis of gene expression? Assume we want to examine the difference in gene expression between normal and tumor cells. We would isolate the RNA from both types of cells and subject it to microarray analysis.
Image from here.
The result will show you how the pattern of gene expression is different between the two cell types. Some genes might show higher or lower levels of expression in cancer cells compared to normal cells.
Warning: DNA microarray analysis will give you so much data that your eyes just might glaze over. Your biggest challenge will become where to actually begin sifting through all the data.
If wondering how a Southern blot is actually performed keeps you up at night, never fear! Here's a clip from The X-Files where Scully performs a Hollywood-style Southern blot. Enjoy the bonus creepy music.