Despite the fact that biochemical study requires the destruction of the cell’s anatomy, gentle fractionation methods have been devised to separate the different cell components while preserving their functions. With these amazing procedures, you can divide a cell into its working organelles and macromolecules in the same way you can split a tissue into its live constituent cell types.

Cell fractionation is a technique for separating cellular components while keeping their distinct functions. This technique initially showed where various biochemical activities occur within the cell.

Subcellular fractionation produces an enriched supply of protein for further purification and aid in detecting certain diseases.

Cell fractionation allows you to study the many components of a cell separately. For example, you can see which organelles produce the most energy after the organelles are isolated.

Let’s take a deeper look into the processes and steps involved in cell fractionation below:

Extraction

Extraction is the first stage in the process of separating any subcellular structures that may exist. To ensure that organelles and biomolecules retain their biological function under mild conditions (known as cell-free systems) before being used, you have to maintain isotopic constraints.

At 0-40°C, the cells or tissues suspend in a solution with an adequate pH and salt content, typically an isotonic solution of sucrose (0.25 mol/L) with a pH of 7.0.

Homogenization/ Breaking The Cells

To get to the information contained within anything, you must first open it. The same is true for cells. You can achieve homogenization through either of the following:

• Grinding
• High pressure
• Osmotic shock

Detergents allow the cell membrane to open up, allowing access to the contents within the cell. Because detergents can interact with both membranes and elements of the cell that are soluble in water, they disrupt cellular membranes. 

In addition, detergents allow cellular components to be mixed or homogenized because they interact with both lipid (membrane) and soluble (cytoplasmic) elements of the cell.

A cell homogenate, or cell lysate, is created when the cellular components are mashed together. In truth, detergents and soaps remove oil and grease by allowing substances that do not ordinarily combine with water to dissolve in water and rinsed away.

In most cases, lysing or opening the cells in detergent is supplemented with a physical approach that further breaks the cell, such as blender-like devices, glass beads, or breaking the cell with sound energy.

Physical methods combined with detergents ensure that all cells in the sample break, allowing you to isolate as much of your cellular fraction as feasible. Modern Biology Inc. can provide you with experiment kits and lesson plans to put these concepts into practice.

Separation

Centrifugation is a technique for separating cell homogenates into fractions by spinning them at high speeds for an extended period. If you have ever been on an anti-gravity carnival ride, you will better understand how centrifugation works. These rides gradually accelerate to the point that you are forced against the walls and no longer require the use of a seat belt to stay upright. 

In a similar vein, centrifugation generates forces that are hundreds of times greater than those produced by gravity, forcing cellular components to the bottom of the container in which they are.

A preparative ultracentrifuge separates (fractionate) the various components of the homogenate through a series of centrifugation. A metal rotor with cylindrical holes to accommodate centrifuge tubes and a motor that spins the rotor fast enough to generate centrifugal forces make up the ultracentrifuge. Theodor Svedberg (1926) invented the ultracentrifuge, which he used to calculate hemoglobin’s molecular weight.

Even small molecules like t-RNA and enzymes can sediment and separate from other components in today’s ultracentrifuges, which rotate at speeds up to 80,000 rpm (rpm=rotations per minute) and exert a gravitational pull of around 500,000 g. It is paramount to keep the ultracentrifuge chamber at a high vacuum to eliminate friction, prevent heating, and keep the sample at 0-4°C.

The determinant of the rate at which each component settles down during centrifugation is by its size and shape and is expressed in terms of the sedimentation coefficient, also known as the Svedberg unit or S-value, where IS = 1 x 10-13 seconds.

Modern Day application of cell Fractionation

From the above information, you have learned what cell fractionation is and how to perform the experiments. However, you need to know how this particular science affects our lives and whether or not it adds value to our general well-being. So, what are some applications of cell fractionation?

Decipher the Molecular Details of Complex Cellular Processes.

The ultracentrifuge has greatly aided our understanding of the functions of many cellular components by isolating organelles and other significant subcellular components. 

Experiments on centrifuge-purified mitochondria and chloroplasts, for example, revealed the fundamental role of these organelles in transforming energy into forms that the cell can use. Similarly, resealed vesicles (microsomes) produced from rough and smooth endoplasmic reticulum fragments have been isolated and examined as functional models of these compartments of the complete cell.

Using purified cell-free systems, scientists can extend this approach to examine many different biological processes free of all complicated side reactions in a living cell. They fractionate cell homogenates in this scenario to cleanse each of the constituents of macromolecules required to catalyze a biological process.

For example, experiments that focused on a cell homogenate that enabled RNA translation to create proteins were used to learn the mechanics of protein synthesis. 

Step by step, fractionation of this homogenate produced ribosomes, tRNAs, and other enzymes, which collectively make up the protein-synthetic machinery. 

You could add individual pure components or withhold one at a time to determine their particular role in the overall process. The restoration of every biological activity in a purified cell-free environment to characterize all its components and action mechanisms is crucial today.

Protein Fractionation

The most common method for fractionating proteins is column chromatography, which involves passing a protein solution down the column containing a porous solid matrix. The interaction of the different proteins with the matrix slows them down to varying degrees, and they can be collected as they flow out of the bottom of the column.

Proteins can be separated based on their charge (ion-exchange chromatography), hydrophobicity (hydrophobic chromatography), size (gel-filtration chromatography), or ability to bind to specific small molecules or other macromolecules, depending on the matrix used (affinity chromatography).

Commercially accessible matrices come in a variety of shapes and sizes. Proteins separate according to the distribution of charges on their surface using ion-exchange columns, packed with tiny beads with either a positive or negative charge. Proteins with exposed hydrophobic regions are slow in hydrophobic columns filled with beads from which hydrophobic side chains protrude.

 Gel-filtration columns, which separate proteins based on their size, are with tiny, porous beads: molecules small enough to pass through the pores linger inside successive dots as they pass down the column. Larger molecules remain in the solution flowing between the beads and thus move more quickly, emerging first from the column.

Therefore, Gel-filtration chromatography is a convenient approach to determine the size of molecules and separating them.

You can perform biochemical analysis of cell populations by disrupting them and fractionating their contents using ultracentrifugation. Further fractionations enable functional cell-free systems, which are necessary for determining the molecular intricacies of complicated biological processes. You can use this method to investigate protein synthesis,

DNA replication, RNA splicing, the cell cycle, mitosis, and many types of intracellular transport. SDS polyacrylamide-gel electrophoresis can detect the molecular weight and subunit composition of even tiny amounts of a protein.

Proteins are resolved as discrete spots in two-dimensional gel electrophoresis by isoelectric focusing in one dimension, followed by SDS polyacrylamide-gel electrophoresis in the other. Thus, even proteins that are ordinarily insoluble in water can be separated using electrophoresis.

Column chromatography can purify the main proteins insoluble cell extracts; physiologically active proteins can be separated based on their molecular weight, hydrophobicity, charge properties, or affinity for other molecules, depending on the type of column matrix. 

In a typical purification, the sample is passed through a series of columns in order, with the enriched fractions from one column applied to the next. Thus, scientists can now study the biological functions of a protein in-depth once it is purified. In addition, you can quickly determine the masses of proteins and peptides produced from them, causing mass spectrometry. With this knowledge, may use genome databases to deduce the protein’s remaining amino acid sequence from the nucleotide sequence of its gene.

Learn More About Cell Fractionation

Much of what we know about cellular, molecular biology has come from researching cell-free systems. Such studies figure out the molecular aspects of DNA replication and transcription, RNA splicing, protein translation, muscle contraction, and particle transport through microtubules, to name a few examples. 

Cell-free systems are used to study the cell division cycle, chromosome separation on the mitotic spindle, and the vesicular-transport processes involved in the transfer of proteins from the endoplasmic reticulum via the Golgi apparatus to the plasma membrane. In theory, cell homogenates also serve as the beginning material for completely separating all the cell’s separate macromolecular components.

Check out the B1-1 and B1-1F!

Deoxyribonucleic acid, abbreviated as DNA, is the hereditary material present in almost all prokaryotes and eukaryotes. DNA is located at the cell’s nucleus, but there is some DNA present in the mitochondria known as mitochondrial DNA. Thus, every cell circulating in your body has the same type of DNA composition. 

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Biological pigments, also known as biochromes or pigments, refer to the substances or molecules that contain color because of selective color absorption. They often operate by absorbing specific wavelengths of light. As a result, the pigment color usually differs from the structural color. Whereas the pigment color is the same from all the angles, the structural color is an outcome of selective iridescence or reflection. There are usually two types of biological pigments: flower pigments and plant pigments. The color you see in flowers is because of the flower pigment. Plant pigments are responsible for controlling growth, photosynthesis, and development.

How Do Pigments in Plants Work?

Plant Pigments work by absorption of various wavelengths of light. Light is the primary source of energy and the most important environmental factor for all plants. For a plant to survive, specifically in the photosynthesis process, it needs to be absorbent to photoreceptor-treated pigments.

The measure of the wavelength of radiation that a pigment can absorb is known as the absorption spectrum. The type of pigmentation is determined by selective absorption of different wavelengths. For example, in higher plants, chlorophyll absorbs blue and red wavelengths and not green wavelengths, and this gives their leaves the green color characteristics.

The pigment’s molecular structure determines the absorption spectrum of each pigment. In this case, a pigment molecule may absorb some wavelengths and not others. This is because the molecular structure of the pigment has limited energy states that it can absorb.

Once a pigment has absorbed some wavelengths and is rejuvenated to a higher energy state, it can utilize the energy in three of the following possible ways:

1. It can discharge it as heat.
2. It can discharge it as radiation of a longer wavelength (lower energy).
3. The energy can be used in photochemical work, i.e., initiation of chemical change.

Betalains, carotenoids, and Flavonoids are the common plant pigments known to emit most of their absorbed light energy as heat. Other plant pigments such as chlorophyll, rhodopsin, phycobilin, and phytochrome use much of their absorbed energy to produce various chemical changes within the plant.

It is important to note that each plant pigment reacts with a narrow range of the spectrum. Thus, there is a need to produce several other pigments with different colors to capture more sunlight energy.

Principal Pigments Explained

Plants have several pigment molecules, far more than animals. It’s because plants are creatures of light. They utilize their sense of light to manage their development, growth, and rapid response to environmental changes. Importantly, plants use light as their chief source of energy.

Plant pigments have both biological and physiological functions. For example, they can control plants’ growth, development, and photosynthesis and advertise rewards for animals that disperse seeds and pollinate flowers. There are four main types of plant pigments. Their production or retention determines their molecules, the color of leaves they fall from, and the chemical formula that describes the different atoms that make up the molecule. The four types of pigments in plants include Chlorophyll, Carotenoids, Anthocyanins, Betalains.

Chlorophyll

Chlorophyll is the green photosynthetic pigment in plants and used for photosynthesis. It is produced in the chloroplasts in the photosynthetic tissues of the leaves and occurs in algae, photosynthetic bacteria, and plants.

There are ten types of chlorophyll—chlorophyll a, b, c, d, and e, bacteriochlorophyll a, b, c, d, and e, and bacterioviridin. However, only two types of chlorophyll – chlorophyll a and chlorophyll b are more diverse in higher plants and algae. The pigment drives the process of photosynthesis, which is the process by which plants make their food. Chlorophyll reflects green light and absorbs blue and red light most strongly.

Chlorophyll is an essential pigment in plants that uses light energy to synthesize carbohydrates. All living organisms depend on photosynthesis, either directly or indirectly. Other benefits of chlorophyll include:

• Conversion of sunlight energy into chemical energy.
• Giving plants a green coloration.
• Trapping sunlight, thus allowing easy and free transfer of electrons to carbon dioxide molecules.
• Absorption of energy which then used to transform carbon dioxide and water into carbohydrates and oxygen.
• Absorption of water and other soluble mineral salts from the soil.

The various types of chlorophyll absorb distinct wavelengths of light. The difference in absorption spectra allows a more significant portion of the solar spectrum during photosynthesis. Chlorophyll-a is common in higher plants, cyanobacteria, chloroxybacteria, and algae. Some groups of algae and higher plants also have chlorophyll-b. Several other algae groups contain chlorophyll-c and chlorophyll-d, and towards the end of the leaf’s lifespan, chlorophyll breaks down into nitrogen which the plant reabsorbs.

Carotenoids

Carotenoids are very long-chain and water-repelling plant pigments that are synthesized by many bacteria, fungi, and plants. In plants, carotenoids are in fruits, flowers, leaves, stems, and roots. Within the plant cell, carotenoids are in the plastids membrane. Chloroplasts are the most common examples of plastids that store carotenoids as well as perform photosynthesis.

Carotenoids are often red, yellow, or orange and include a familiar compound, carotene, which gives carrots their color. Beta-carotene, produced by the ray flowers’ chromoplasts, is also used in the sunflower flower plant and gives it its bright yellow-orange colors. Beta-carotene also gives other vegetables and sweet potatoes an orange color. Lycopene is another type of carotenoid pigment that gives tomatoes their red color.

The carotenoids work by absorbing the blue wavelengths and allowing the longer wavelengths to be scattered, producing the yellow color.

There are two important benefits of carotenoids in plants. Firstly, they help in photosynthesis. Carotenoid pigment helps transfer the sunlight they receive to chlorophyll, which uses it in photosynthesis. Secondly, carotenoids help in the protection of plants that are over-exposed to sunlight. This is because they dissipate excess light energy, which is absorbed as heat.

Excess light energy can destroy the protein membrane and other molecules in the absence of carotenoids. Other important functions of carotenoids include making fruits and flowers conspicuous to animals for dispersal and pollination. In addition, Beta-carotene is a good source of vitamin A in animals.

Anthocyanins

Anthocyanins are dispersed and water-soluble pigments in vacuoles, membrane-enclosed structures inside the cells that store nutrients and water. Depending on the PH, anthocyanins may appear purple, red, blue, or black. The pigments are found in fruits, flowers, and vegetables. Plants and fruits like black soybean, blueberry, black rice, raspberry, apples, roses, and wine are some of the foods rich in anthocyanins. In addition, anthocyanins are responsible for the red leaves during autumn.

Anthocyanins are synthesized through the phenylpropanoid pathway and belong to the molecule class of flavonoids. In higher plants, they often occur in fruit where they attract animals that eat fruits and disperse seeds helping in pollination. They can also be found in flowers, where they attract insect pollinators.

Although they color beverages and foods, anthocyanins have not yet been approved to be used as food additives or supplement ingredients. Some of the major benefits of anthocyanins in plants include:

Provide coloration

Light absorbance

Betalains

Betalains are nitrogen-containing and water-soluble vacuolar pigments. They are characterized by red or yellow color and contain betaxanthins and betacyanins and are used as color additives in food. The pigments are often noticeable in flower petals and can also be found in leaves, fruits, roots, and stems of plants containing them. There two types of betacyanins:

• Betacyanins: Features the reddish to violet Betalains pigments. Some of the plants that are rich in betacyanins include neobetanin, probetanin, betanin, and isobetanin.
• Betaxanthins: Features yellow to orange Betalains pigments. Some of the common plants with betaxanthins include indicaxanthin, vulgaxanthin, portulaxanthin, and miraxanthin.
The key benefits of Betalains in plants include:
• The visual attraction for seed dispersers and pollinators: The red or/and yellow color of the Betalains may attract a variety of herbivorous animals and insects, which may help in pollination and seed dispersal.
• Provide anti-inflammatory, antioxidant, and detoxification support. Research has also shown that Betalains lessens the tumor cell growth and minimizes the risk of cancer.
• The red or/and yellow pigments can also protect plants from herbivores attracted by the green color.

Biology Experiments to Teach this to Your Students

Modern Biology offers you exciting biological lessons geared to help you understand more about the different aspects of plant and animal life. The platform provides multi-lab programs to its students, cutting across from understanding the primary function of pigments to other complex concepts and practical experiments ranging from high school biology to molecular biology and genetics.

For questions or concerns pertaining to biological lab courses and experiments, reach out to us at (765) 446-4220 or fill out our online form today.

Just like people communicate through words, the cells that make up our bodies have a way of communicating to perform the different functions and processes that keep the body alive and responsive.

But of course, there are no talking molecules in the world of cells—they have to use cell receptors and signaling sequences to pass information from one cellular region to another. This is achieved through receptors (receiving molecules) and ligands (signaling molecules). 

Receptors and ligands come in various forms, but they always have one trait: they come in pairs, with a receptor recognizing just one (or a few) particular ligands and a ligand attaching to only one (or a few) target receptors. When a ligand binds to a receptor, it alters its shape or function, enabling it to send a signal or cause a change within the cell.

One important component that allows this type of signaling is the cell surface receptor. Let’s look at what exactly cell surface receptors are and their role in converting information from outside the cell into a change within the cell.

In a nutshell, cell surface receptors also known as transmembrane receptors are membrane-linked proteins that bind to ligands (signaling molecules) outside the cell surface and perform signal transduction by converting extracellular signals into intracellular signals.

The ligand doesn’t have to penetrate the plasma membrane in this kind of signaling. As a result, ligands may be a wide range of molecules from large to hydrophilic (water-loving) compounds. A typical cell-surface receptor contains three domains or protein regions: 

• An extracellular domain (outside the cell) ligand-binding domain,
• A hydrophobic domain that extends through the membrane
• An intracellular domain (inside the cell) which often sends a signal.

Signal Transduction

A series of biochemical changes that occur inside the cell or the alteration of the cell membrane potential by the flow of ions in and out of the cell sustain signal transmission. Receptors that cause biochemical changes may either directly or indirectly activate intracellular messenger molecules via intrinsic enzymatic activity inside the receptor. The transduction process requires four parts:

• Extracellular signaling molecule: Produced at one cell but can move from cell to cell
• Intracellular signaling proteins: Transmit the signal to the cell components
• Target proteins: Altered when an active signaling pathway and changes the behaviors of a cell
• Receptor protein: Bind to the signaling molecule and help pass information into the cell

The activation and/or inhibition of signaling cascades that control various cellular activities, such as cell growth, proliferation, survival, and invasion, are triggered by ligand binding and cell surface and internal receptors’ activation. The diagram below represents a signal transduction pathway.

The Three Types of Cell Surface Receptors

The hydrophobic region may consist of several stretches of amino acids that crisscross the membrane, and the size and structure of these sections may vary greatly depending on the kind of receptor. There are many kinds of cell surface receptors, but at this time, we’ll look at the three primary receptors: Ion channel-linked receptors, G protein-linked receptors, and Enzyme-linked receptors. 

Ion Channel-Linked Receptor

The neurotransmitter binds to the receptor and changes the protein structure during signal transduction in a neuron. This allows extracellular ions to enter the cell by opening the ion channel. As a result, the plasma membrane’s ion permeability is altered, converting the extracellular chemical signal into an intracellular electric signal that affects cell excitability.

The acetylcholine receptor is a cation channel-linked receptor. There are four subunits in the protein: alpha (α), beta (β), gamma (γ), and delta (δ). There are two alpha (α) subunits, each of which has an acetylcholine binding site. Moreover, there are three possible conformations for this receptor. The native protein conformation is closed and unoccupied. 

When two acetylcholine molecules attach to the binding sites on alpha (α) subunits, the receptor’s conformation is changed, and the gate is opened, enabling numerous ions and tiny molecules to pass through. However, this open and occupied state only lasts a short time after which the gate is closed, resulting in the closed and occupied state. In this case, the two acetylcholine molecules dissociate from the receptor, restoring it to its original closed and unoccupied condition.

G-Protein-Linked Receptors

The G-protein is a trimeric protein made up of three subunits designated as alpha (α), beta (β), gamma (γ). The alpha (α) subunit releases bound guanosine diphosphate (GDP) in response to receptor activation, which is then displaced by guanosine triphosphate (GTP), resulting in the activating the subunit, which subsequently dissociates from the β, and γ subunits, and the cycle starts all over again. The activated subunit may also have a direct effect on intracellular signaling proteins or target functional proteins.

In eukaryotes, G-protein-linked receptors (GPCRs) are the most numerous and varied category of membrane receptors. Light energy, peptides, lipids, carbohydrates, and proteins all pass via these cell surface receptors, which serve as an inbox for communications. Cells receive these signals to notify them of the presence or absence of life-sustaining light or nutrients in their surroundings or to relay information from other cells.

GPCRs are involved in a wide range of activities in the human body, and a better knowledge of these receptors has had a significant impact on contemporary medicine. In fact, experts believe that GPCRs are involved in the action of one-third to half of all marketed drugs.

Photosensitive chemicals, odors, pheromones, hormones, and neurotransmitters are examples of ligands that bind and activate the G-protein-linked receptors, and they vary in size from small molecules to peptides and large proteins. The cAMP signaling pathway and the phosphatidylinositol signaling pathway are the two main transduction pathways involving G-protein coupled receptors. Both are mediated by the activation of G proteins.

Enzyme-Linked Receptors

Enzyme-Linked Receptors (mainly receptor tyrosine kinases) are Cell-surface receptors with intracellular domains connected to an enzyme. In certain instances, the receptor’s intracellular domain is an enzyme, and in some, the enzyme-linked receptor’s intracellular domain interacts directly with an enzyme. Examples of enzyme-linked receptors include: 

• Receptor tyrosine kinases (RTKs), 
• Receptor serine/threonine kinases, 
• Receptor-like tyrosine phosphatases, 
• Histidine kinase-associated receptors, and 
• Receptor guanylyl cyclases

Receptor tyrosine kinases are the most common and have the most applications. Growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), and hormones such as insulin bind to the majority of these molecules.

After binding with their ligands, most of these receptors will dimerize in order to trigger further signal transductions. When the epidermal growth factor (EGF) receptor binds to its ligand EGF, the two receptors dimerize, and the tyrosine residues in the enzyme part of each receptor molecule are phosphorylated. This activates the tyrosine kinase, which then catalyzes further intracellular processes.

Receptor tyrosine kinases can be broken up into seven categories. Each subgroup is represented by just one or two members. In certain subfamilies, the tyrosine kinase domain is interrupted by a “kinase insert region.” The functions of the majority of cysteine-rich, immunoglobulin-like, and fibronectin-type III-like domains are not yet known.

Phosphate groups are removed from phosphotyrosine residues by receptor-tyrosine phosphatases, which counteract the actions of receptor-tyrosine kinases. Receptor tyrosine phosphatases, in many instances, act as negative regulators in cell signaling pathways, ending signals started by protein-tyrosine phosphorylation. On the other hand, some protein-tyrosine phosphatases are cell surface receptors whose enzymatic activities aid cell signaling. 

A receptor called CD45, which is expressed on the surface of T and B cells, is an excellent example. CD45 is believed to dephosphorylate a particular phosphotyrosine after antigen stimulation, inhibiting the enzymatic activity of Src family members. As a result, the CD45 receptor-tyrosine phosphatase stimulates nonreceptor protein-tyrosine kinases, which is rather paradoxical.

The extracellular and intracellular domains of enzyme-linked receptors are typically extensive, but the membrane-spanning portion is made up of one alpha-helical peptide strand. When a ligand attaches to the extracellular domain, a signal is sent across the membrane, activating the enzyme, which puts in motion a series of processes inside the cell that ultimately results in a cellular response.

Cell disorders such as cancer are defined by anomalies in cell growth, proliferation, differentiation, survival, migration, and anomalies in signaling through enzyme-linked receptors. This is evidence that Enzyme-linked receptors play a significant role in the development of this class of illnesses. 

There are many lab experiments that can be conducted to observe the cell surface structure and help you familiarize yourself with the current strategies used in the study of cell surface receptors and other important regulatory molecules. At Modern Biology Inc, we have high-quality educational products and experiments to help students and tutors when learning about cell surface receptors and other important biological concepts. For more information, reach out to us at (765) 446-4220 or fill out our online form, and one of our team members will get back to you in a jiffy.

The polymerase chain reaction, more commonly referred to as PCR, is sometimes explained as “molecular photocopying.” PCR is an inexpensive and convenient way to copy or “amplify” small chains of DNA into samples larger enough for molecular and genetic analyses.

PCR uses relatively short DNA sequences known as primers to choose the portion of the genome to be amplified. The sample is buffered in a saline solution to keep pH constant, and DNA-replicating enzymes are added. The temperature of the mixture is raised and lowered repeatedly over a period of several hours to assist a DNA replication enzyme in copying the primer. The result is up to a billion copies of the prime DNA sequence in just a few hours.

Once a strand of DNA has been amplified by PCR, the product can be used in a variety of laboratory procedures. PCR is a first step in testing for many kinds of bacteria and viruses, including HIV and COVID-19. Amplified DNA is useful in the detection of many genetic disorders. And the human genome project would not have been possible without PCR.

Prior attempts to decode the human genome had relied on techniques that would begin at one telomere of a strand of DNA and attempt to read millions of bases until it reached the telomere at the other end. With PCR, however, the human genome project sequenced multiple strands of DNA at different places but at the same time. The cost of reading the human genome was reduced by a factor of over 100,000 and sped up by years.

There are a huge number of applications of PCR beyond the human genome project. PCR might be used by a researcher who wants to study a single gene. Or maybe a crime scene detective is looking for a genetic marker to rule out a suspect or justify further investigation. Or an ancestry service may want to match DNA sequences between people who don’t know each other to confirm that they are biologically related.

PCR is widely regarded as one of the most important advances in the study of molecular biology in the twentieth century. Its creator, Kary B. Mullis, was awarded the Nobel Prize for Chemistry in 1993.

PCR Process Steps Explained

The three PCR steps seem almost intuitive since the technique has been so widely used, although the invention of PCR was revolutionary in its time. Before the steps make sense, however, there are two inputs into PCR testing about which we need to give further detail.

Taq Polymerase

Cells have to replicate their DNA with a DNA polymerase before they can divide. The DNA polymerases that power mitosis make new strands of DNA on existing strands of DNA they use as templates.

PCR uses a specialized polymerase known as the Taq polymerase. This polymerase gets its name from the thermophilic bacterium in which it was first isolated, Thermus aquaticus. This bacterium lives in thermal vents of underwater volcanoes. It survives at temperatures that would denature proteins and enzymes in most other species. Its polymerase is optimally active at temperatures around 70° C, or 158° F for your students.

Heat stability is what makes the Taq polymerase the ideal choice for PCR. This procedure makes repeated use of high temperatures to denature and separate the strands of DNA as they are replicated.

PCR Primers

Polymerases, including Taq polymerase, can only make DNA if they are given a template, the primer we mentioned previously. Every polymerase uses a short sequence of A-C-G-T nucleotides as a starting point. The experimenter chooses the primers that will be amplified by the Taq polymerase in PCR.

Primers are very short chains of DNA, usually around just 20 nucleotides long. Every PCR reaction uses two primers chosen to flank the target region to be copied. This way the primer bind to opposite strands of DNA, at both edges of the segment the experimenter seeks to copy. The primers then bind to the DNA template by pairing complementary bases. An A-G-C-T-A-G-C-T-A-G-C-T-A-G-C-T template would bond to a T-C-G-A-T-C-G-A-T-C-G-A-T-C-G-A primer.

Once the primers bind to the template, they are extended by the Taq polymerase, and the nucleotides between them will be copied.

Those basics out of the way, we can now talk about the 3 steps of PCR.

3 Steps of PCR

The essentials of PCR are the Taq polymerase, template DA, primers, and the A-G-C-T nucleotides. The experimenter assembles these ingredients in a tube, along with the cofactors that regulate the kinetics of the Taq enzyme, and puts the mixture through repeated heating and cooling that power the synthesis of DNA.
The three basic steps are:

1. Denaturation at 96° C, heating the polymerase, DNA to be copied, primers, and nucleotides so strongly that the sample DNA is denatured. Its strands unravel. Denaturation converts double-stranded DNA to single-stranded DNA for the next step.
2. Annealing at 55 to 65° C cools the reaction, so the priers can bind to their complementary nucleotide sequences
3. Extension at 72° C raises the reaction temperature so Taq is active, extending the primers, and synthesizing new strands of DNA.

PCR repeats this cycle 25 to 35 times over a typical reaction that takes two to four hours. Copying shorter segments of DNA takes less time, and copying longer segments of DNA takes more time. Efficient PCR reactions can create billions of copies of DNA, because it’s not just the original DNA that is used as a template each time. Every copy of DNA serves as a template for the next round of synthesis.

The number of DNA molecules roughly doubles with each reaction. As a result, 25 rounds of PCR can create 2 25 or over 30 million copies of the DNA being studied, and 35 rounds of PCR can create 235 or over 34 billion copies of the target DNA.

Visualizing PCR Results with Gel Electrophoresis

The next step in characterizing DNA (usually) is visualizing the results of PCR with gel electrophoresis. As students of teachers who use Modern Biology’s gel electrophoresis supplies and all-in-one gel electrophoresis laboratory teaching kits are well aware, gel electrophoresis is a technique that uses electrostatic charge to pull segments of DNA through a gel matrix. DNA is dyed with ethidium bromide, so it glows under UV light. Experimental and control samples of DNA are placed at one end of a buffered gel (buffered with saline to keep pH stable) in wells covered by the conductive gel.

DNA has a slight negative charge, so DNA samples are placed nearest the cathode, the negative charge attached to the gel. They are pulled toward the anode, the positive charge at the other side of the gel. Shorter segments get pulled faster than longer segments, so when DNA is visible at the anode side of the gel, power is turned off.

If everything has gone as planned, the electrostatic charge has created a “ladder” of DNA samples with known base pair lengths at one side of the gel, and the tested DNA has arrived near or between two controls that give a rough estimate of how many base pairs are in the sample. The length of the DNA in the sample is now estimated, and the millions or billions of copies of test DNA now visible under UV light, can be cut out of the gel for further analysis.

Practical Applications of PCR

One of the questions that comes up in biology classes at all levels since the beginning of the pandemic is “What is a COVID-19 test, anyway?” The answer is, although there are several approaches to COVID-19 detection, the most commonly used method is a highly automated polymerase chain reaction test.

Real-time PCR amplifies a known primer of COVID-19 DNA that does not carry the risk of causing disease. Amplifying this segment of DNA tens of millions of times, that is, if it is to be found on the swab, automated detection equipment can detect COVID-19 with 97-percent specificity and 100-percent sensitivity. Chances are that many of your students will have had PCR testing.

PCR, of course, has many other applications that pique student interest. PCR can be used to amplify fetal DNA from a tiny sample of amniotic fluid to test for genetic issues. PCR can amplify bacterial or viral DNA to test for the presence of pathogens in a patient’s bone sample, skin sample, serum, or spinal fluid.

PCR is of enormous value in forensics. The DNA in a single hair follicle can be amplified for testing that can exclude an innocent suspect or point to the need for further investigation to establish probable cause for an arrest.

Modern Biology provides everything busy biology instructors need to introduce their students to hands-on experiences of PCR. Our experiments aren’t just demonstrations of PCR. Your students will use PCR to confirm or disconfirm a scientific hypothesis of their own making. And if you want to go beyond our experiments, we have all the PCR supplies you need.

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