Section 1.1: Analyzing Biological Molecules

In earlier discussions, we explored essential branches of biology, such as molecular biology, biochemistry, and genetics. A recurring theme involved simplifying the complexity of biological systems to focus on key components, like DNA, proteins, and genetic inheritance.

We discovered methods to isolate specific biochemicals. But what follows after obtaining a relatively pure sample? One fundamental approach to analyzing these biochemicals is through size differentiation. Many biochemicals consist of long chains of monomers; for instance, DNA and RNA comprise long sequences of four types of nucleotides, while proteins are formed from 20 different amino acids. The length of these chains is a physical characteristic that enables us to separate purified DNA or protein samples.

Now, let’s delve into DNA analysis.

Section 1.2: Agarose Gel for DNA Analysis

To visualize this process, picture a mixture of silt, gravel, and stones. If you wish to segregate them based on size, you could create a layered mesh of screens, starting with coarser materials at the top and progressing to finer ones at the bottom. As you pour the mixture at the top, gravity (possibly aided by agitation or water) would help the materials navigate through the screens.

In the end, the largest rocks are filtered by the coarsest screen, while smaller particles progressively pass through to the smaller screens, with the finest materials settling at the bottom.

This principle is analogous to how we handle biochemicals, albeit with some vital differences. For our purposes, think of the DNA mixture as a colander filled with cooked spaghetti at a molecular level. Additionally, DNA carries an electrical charge due to phosphate groups between nucleotides, which possess a negative charge. This charge increases with the length of the DNA strand, a fact that will become significant shortly.

Instead of using various-sized screens, we will create a gel—a consistent three-dimensional mesh formed by cross-linking a complex polymer. Agarose, a polysaccharide derived from seaweed, serves as an economical and reliable gelling agent. It has been utilized for centuries, often seen in agar plates for microbial growth, which consist of agarose mixed with smaller sugars known as agaropectins.

Creating an agarose gel is straightforward, akin to making jello. A 1% agarose gel typically requires 1 gram of agarose powder dissolved in 100 milliliters of buffer. A commonly used buffer for DNA gels is TAE, which will be discussed further. Agarose dissolves in water or buffer at near-boiling temperatures and solidifies at around 34–38 degrees Celsius.

After microwaving and dissolving the agarose, we pour it into a tray or mold, inserting a comb to form evenly spaced wells for loading DNA samples.

Section 1.3: Agarose Gel Electrophoresis

Electrophoresis is a technique that utilizes electric charge to propel molecules through a supportive medium—in our case, the agarose gel. Here, electric charge supplants gravity as the driving force for separation, with DNA migrating towards the positive electrode.

After preparing the gel, we need to sort DNA by length. The gel is placed in an electrophoresis chamber filled with a buffer that maintains a stable pH throughout the procedure. The DNA samples are loaded into the wells at one end of the gel. A voltage is applied, with a negative electrode at the sample end and a positive one at the opposite end.

Due to their negative charge, longer DNA strands will migrate towards the positive electrode, while smaller strands will move faster through the constant-sized pores in the gel. The experiment continues until the smaller DNA fragments have traveled a considerable distance, at which point the voltage is halted. The DNA's position is visualized using specific dyes that bind to DNA and fluoresce.

Let’s examine the practical steps in detail.

This video provides an introduction to the principles and practical aspects of electrophoresis, including the support medium and buffer used.

Section 1.4: Setting Up the Electrophoresis Apparatus

The electrophoresis setup consists of a tank filled with buffer, housing the tray containing the agarose gel, where electrodes apply a fixed voltage.

The buffer serves dual purposes: it provides ions to conduct current and maintains a stable pH. A standard buffer for DNA analysis is TAE, which stands for Tris base, acetic acid, and EDTA. The Tris and acetic acid create a buffer typically set at pH 8.3, while EDTA protects DNA from degrading enzymes by capturing divalent ions.

Here’s a step-by-step protocol for preparing TAE buffer: 1. Fill the tank with TAE. 2. Place the agarose gel into the tank.

Next, we prepare the DNA sample, which cannot be placed into the wells alone. Instead, it needs to be mixed with a sample loading buffer containing a dense substance like glycerol or sucrose to ensure the DNA sinks to the well's bottom. The loading buffer also includes dyes such as bromophenol blue to track the DNA's migration through the gel.

After mixing the DNA with the loading buffer, we pipette the samples into the wells.

The power supply applies a constant voltage during the experiment—typically between 1 and 5 volts per centimeter of distance between electrodes. As the DNA moves through the gel, we monitor the progress using the dye's position.

Visualizing the DNA's location in the gel is straightforward. Ethidium bromide, a common intercalating dye, can be added either before or after running the gel. When exposed to UV light, this dye fluoresces bright orange, allowing for easy detection of DNA.

This video illustrates the gel electrophoresis process, showing the steps involved in analyzing DNA samples.