Good morning, everyone! Welcome to the first Biology for Writers post of the new year! Today, I wanted to talk about some common techniques used in genetics and molecular biology, particularly those I use in the lab, since those are what I’m most familiar with. As a result, the list will probably be skewed toward plant biology, but I will include other techniques I’m somewhat familiar with too. If you’re interested in a specific discipline or something you don’t see listed here, let me know in the comments, and I’ll be happy to tell you what I know or help you find some resources! Let’s get started!
Why? If you’re working in genetics or molecular biology, you often need to isolate a nucleic acid, be it DNA or RNA. These molecules are useful for learning about genes and the various proteins, including enzymes, they produce (more on this another time).
Why DNA vs. RNA? Well, the DNA contains a gene’s complete sequence, including introns (which are removed in the messenger RNA that determines the protein’s amino acid sequence) and regulatory sequences that control when the gene is turned on or off. So if you’re interested in the pure molecular genetics of an enzyme, you might extract DNA. DNA is also useful if you’re trying to sequence a new genome (the complete DNA of an organism); genomes are often useful for future research.
RNA, on the other hand, might be useful if you want to, say, produce a human protein in bacteria. To do this requires gene cloning, which is much easier with only the coding sequence (the part that actually codes for the protein–i.e. messenger RNA) rather than all the extra introns and “junk DNA” that comes along with a complete gene. So for things like genetic engineering, it’s better to extract RNA and back-convert it into DNA (more on this in a minute).
How? Extracting DNA/RNA is, in a nutshell, getting it out of the cells of your plant or bacteria or whatever organism you’re starting with, and separating it from the proteins, fatty acids, and other stuff that also belongs in a cell. (If extracting DNA, you also want to separate it from RNA, and vice versa.) The first thing to do, then, is to break open your cells. In plants, you usually start this by freezing your leaves in liquid nitrogen, which helps minimize molecular degradation, and grinding them up in a mortar and pestle. Then, a lysis (cell-opening) buffer is added to fully lyse the cells. (Buffers are solutions with a specific pH that “buffer” against big changes in acidity.) During this process, nucleases, which degrade DNA, are inhibited by the cold temperature from the liquid nitrogen. Other chemical inhibitors may be added as well.
Next, a series of centrifuging steps separates the DNA (in various liquid buffers) from proteins, lipids, and other cellular detritus (often in the form of a pellet at the bottom of the little test tube). DNA in the top liquid, or “supernatant,” is removed into several different tubes, until ultimately it is collected without the liquid on a little pad within a special tube. The DNA is then resuspended, or “eluted,” in super-sterile water (a small amount, like 150 microliters–a microliter is a millionth of a liter). This serves to make the DNA more concentrated. You can then measure how much DNA you extracted with an instrument called a fluorometer, and if you got enough DNA, you’re all set to go on to the next step!
Polymerase Chain Reaction
Why? Once you’ve extracted DNA, what do you do with it? If the answer is “isolate a gene,” your next step is polymerase chain reaction (PCR). PCR is an “amplification” technique that takes advantage of DNA’s structure to make many copies of your gene of interest. Then, you can move on to DNA sequencing, genetic engineering, or something else.
How? Let me first make a small digression into the basics of DNA’s structure. DNA is made up of four kinds of nitrogenous bases (let’s just call them A, T, C, and G) that join together in two long strands. The bases pair in a complementary manner to form that familiar double helix shape; A only pairs with T and C with G. When I talk about “DNA sequence,” I’m talking about the order of bases; AATCG is different from ATACG, for example. “Complementary sequence” refers to the sequence of bases on the other strand of the DNA; each DNA strand is the opposite of the other. For example, TTAGC is complementary (opposite) to AATCG. Got it? Great! Let’s go on to the technique.
PCR exploits DNA’s structure to make many, many copies of a gene or other sequence of interest. You can find a great fact sheet about PCR, including a helpful figure, here. Basically, if you know the sequence of your gene of interest, you can order custom short DNA molecules, called oligonucleotide primers (or just primers), of 15-25 base pairs each which are complementary to the ends of your gene. In a tiny little test tube, you mix together DNA, water, primers, and free nucleotides, and stick it all in a machine called a thermocycler, which can be programmed to heat and cool the tube for certain times. The heating profile basically does the PCR.
There are three basic steps to PCR. First, denature the DNA (separate the strands) by heating to about 95 Celsius. Next, drop the temperature to about 60 Celsius for annealing (of primers to DNA). The last step is extension (addition of new nucleotides to make a new DNA molecule), which happens at around 70 Celsius. Together, these steps make one cycle. PCR is usually repeated in about 36 cycles, which because there are more templates with each cycle, makes thousands of copies of the gene of interest, hence “DNA amplification.” PCR is a very useful technique and is indispensable in most genetics laboratories.
In fact, a form of PCR called reverse transcription-PCR (RT-PCR) is used to back-convert extracted RNA into complementary DNA (cDNA). RT-PCR uses primers that are specific for messenger RNAs, enabling the isolation and amplification of only the coding sequence of an organism’s genes. This is the next step from RNA extraction (see above).
Why? This is a sort of confirmation technique. Once you have your extracted DNA/RNA or your PCR products, you want to make sure you did your previous techniques right, so you run an agarose gel. This helps you determine the size of your DNA fragments (or genomic DNA in the case of an extraction), so you know you can proceed with the next step in your research.
How? Gels are made from agarose, a compound found in seaweed, and buffer. You pour molten agarose into a rectangular mold with a comb stuck in, so when it hardens and you pull the comb out, you have a flat, rectangular Jell-O like surface with a line of wells at one end. After pouring buffer over the gel so it’s under the surface of the liquid, you mix your DNA/PCR product with a blue loading dye and put one sample into each well (called a “lane” once you’ve run the gel). You should always load a size standard, called a “ladder,” into one lane, and it’s good to run a positive control (you know the result will be what you want) and negative control (you know the result will be the opposite of what you want), so that if something’s wrong with your samples but not the controls, you know it’s the samples themselves, not your PCR.
Once you’ve loaded all your samples, the next step is to turn on the electric current and let the gel run. Because DNA has a slight negative charge, it will migrate toward the positive end of the gel (away from the wells in which you loaded it). As it moves, it will bump into all sorts of molecules within the gel, i.e., the gel itself will get in the way of the DNA’s movement. This allows for size separation of DNA fragments; smaller fragments will bump into the gel less and therefore move a greater distance in the same time. When you look at the gel under ultraviolet light, you see bright “bands” that indicate where the DNA is; the closer to the wells, the longer the DNA fragment. Comparing these bands to the ladder size standard (which shows up as many bands of known sizes) allows you to identify the size of your PCR product, so you can tell whether you got the right gene. This makes gel electrophoresis an immensely useful technique.