The objectives of this experiment are to extract DNA from a fruit sample, test the moisture of a soil sample, and perform blood typing and gel electrophoresis. The DNA will be extracted using the basic biochemical techniques for isolating, purifying, and digesting DNA molecules. The moisture test will be completed using an Arduino moisture sensor. A blood typing kit will be used for the blood tests. While these tests are performed, the gel electrophoresis will be run simultaneously.
DNA is the blueprint of life and is found in almost all living organisms. These organisms can be as simple as a single-celled bacteria or as complex as a multi-celled human: the human body contains approximately 50 trillion cells. There are two different types of cells: prokaryotes and eukaryotes. An example of prokaryotic organism is bacteria. Prokaryotic cells do not contain a nuclear membrane and so do not have a distinct nucleus. Only eukaryotic cells, which make up plants and animals, will be considered in this lab. Eukaryotic cells have a distinct, membrane-bound nucleus that isolates the DNA from the rest of the cell. The structure of plant cells is different from those of animal cells in structure and cellular contents. Only plant cells will be used in this experiment.
Figure 1: Cell membrane.Plant cells are surrounded by a cell wall. It has high mechanical strength and protects the cell. Directly beneath the cell wall lies the plasma membrane (Figure 1), which contains the cytosol. The various cell organelles, including the nucleus, are found within the cytosol. The nucleus houses the DNA in the form of chromatin.
Figure 2: Cell nucleus.Chromatin is the active form of DNA in the cell when it is not preparing for cell division. It is comprised of DNA wrapped around protein particles called histones.
Figure 3: Cell structure.In this experiment, a goal is to extract the DNA from a fruit sample. Some knowledge of the scientific background behind DNA extraction is needed to do this.
The DNA extraction process is a fairly simple biochemical procedure that can be divided into three major steps: breaking open the cell (lysis), destroying membranes within the cell, and precipitating the DNA out of the solution.
The following sections describe how each step relates to the physical and biochemical properties of DNA.
Plant cells have a very rigid external structure — the cell wall — which protects it. To get to the DNA, the very first step would be to break open that wall.
The cell wall is the first barrier in that must be broken to extract the DNA molecule inside the cell. It is very rigid and acts as a protector and filter. It is made of cellulose, and is responsible for making wood hard and durable. To destroy the cell wall, a mechanical method is used to break apart the cellulose molecules. In this experiment, the fruit sample is mashed manually.
The cell's plasma membrane is made of phospholipid bilayers; they are made of fat. To disrupt them, that mesh of fat molecules is broken up with soap. The structure of soap is very similar to that of fat and grease.
Figure 4: Soap micelle.A soap molecule has two parts: a head and a tail. The head is polar and is attracted to water while the tail is non-polar and is attracted to oil and fat. When soap molecules are in water, they group themselves into micelles — a roughly spherical structure in which all the polar heads point outwards (in contact with water) and all the non-polar tails point inwards at the center of the sphere (away from the water). They can effectively trap the fat molecule inside the micelle and dissolve the cell membranes. How does this micelle break down the phospholipid bilayer? The molecules in the phospholipid bilayer (Figure 5) also contain molecules that are made up of a hydrophobic head and a hydrophilic tail. The soap molecules orient themselves so that their head associates with the tail of the phospholipid bilayer. In this way, the soap is able to break up the bilayer molecule by molecule.
Figure 5: Phospholipid bilayer.When the membrane is successfully disrupted, the DNA is released from the cells into the solution along with protein molecules and other cellular miscellanea.
The DNA molecule is a double-helical polymer consisting of a sugar-phosphate backbone with nitrogenous bases running perpendicular to the backbone. These bases, often represented by letters — A (adenine), G (guanine), C (cytosine), and T (thymine) — are the elementary components making up the coded genetic information (Figure 7). The base sequence acts as the instruction manual of the cell, directing it on how to make proteins and other important molecules that an organism needs to survive and function.
With the cell's contents mixed into a solution, the DNA is separated from the rest; this process is called precipitation. Salt is used because it disrupts the structure of the proteins and carbohydrates found in the solution. Also, the salt provides a favorable environment to extract the DNA by contributing positively charged sodium ions that neutralize the negative charge of DNA. After the addition of salt and soap, the manner by which the DNA is being extracted out of the solution cannot be seen as it is too small to distinguish from the rest of the solution. To aid in precipitating the DNA, alcohol is added since it cannot dissolve DNA. A translucent white substance will begin to form at the top; this is DNA. Once it is thick enough, it can be spooled out. This simple procedure is a rough extraction process that needs further purification before it can be successfully run on a gel for analysis.
Figure 6: Extracted DNA.Often times, larger fragments of DNA are cut, or restricted, to extract a particular fragment. This is made possible by the action of restriction enzymes, which are used by bacteria to cut up foreign or enemy DNA. Restriction enzymes are catalytic proteins that recognize specific palindromic DNA sequences and cut the double-stranded DNA at particular sites. The sites that the restriction enzymes recognize are called restriction sites. There are many different types of restriction enzymes. Each type recognizes a different restriction site. In this lab, Lambda DNA, which is a commercially available DNA normally found in a virus called Phage Lambda, will be restricted with the restriction enzyme BamH1.