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Nucleic Acids: High molecular weight polymers containing a mixture of purine and pyrimidine nucleotides chained together by ribose or deoxyribose linkages.

What are Nucleic Acids?

JoVE 10684

Nucleic acids are long chains of nucleotides linked together by phosphodiester bonds. There are two types of nucleic acids: deoxyribonucleic acid, or DNA, and ribonucleic acid, or RNA. Nucleotides in both DNA and RNA are made up of a sugar, a nitrogen base, and a phosphate molecule.

A cell’s hereditary material is comprised of nucleic acids, which enable living organisms to pass on genetic information from one generation to next. There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA and RNA differ very slightly in their chemical composition, yet play entirely different biological roles. Chemically, nucleic acids are polynucleotides—chains of nucleotides. A nucleotide is composed of three components: a pentose sugar, a nitrogen base, and a phosphate group. The sugar and the base together form a nucleoside. Hence, a nucleotide is sometimes referred to as a nucleoside monophosphate. Each of the three components of a nucleotide plays a key role in the overall assembly of nucleic acids. As the name suggests, a pentose sugar has five carbon atoms, which are labeled 1o, 2o, 3o, 4o, and 5o. The pentose sugar in RNA is ribose, meaning the 2o carbon carries a hydroxyl group. The sugar in DNA is deoxyribose, meaning the 2o

 Core: Macromolecules

Isolating Nucleic Acids from Yeast

JoVE 5096

One of the many advantages to using yeast as a model system is that large quantities of biomacromolecules, including nucleic acids (DNA and RNA), can be purified from the cultured cells.


This video will address the steps required to carry out nucleic acid extraction. We will begin by briefly outlining the growth and harvest, and lysis of yeast cells, which are the initial steps…

 Biology I

Electrophoretic Mobility Shift Assay (EMSA)

JoVE 5694

The electrophoretic mobility shift assay (EMSA) is a biochemical procedure used to elucidate binding between proteins and nucleic acids. In this assay a radiolabeled nucleic acid and test protein are mixed. Binding is determined via gel electrophoresis which separates components based on mass, charge, and conformation.


This video shows the concepts of EMSA and a general procedure, …

 Biochemistry

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

1State Key Laboratory of Reproductive Medicine, Nanjing Medical University, 2The Affiliated Hospital of Hangzhou Normal University, 3School of Life Science and Technology, ShanghaiTech University, 4Department of Tissue and Embryology, School of Basic Medical Sciences, Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University

JoVE 59830

 Genetics

Macromolecules- Concept

JoVE 10590

Biomolecules

Organisms contain a wide variety of organic molecules with numerous functions which depend on the chemical structures and properties of these molecules. All organic molecules contain a carbon backbone and hydrogen atoms. The carbon atom is central in the formation of a vast variety of organic molecules ranging in size, shape and complexity; inorganic molecules on the other…

 Lab Bio

What are Proteins?

JoVE 10677

Proteins are chains of amino acids that are connected by peptide bonds and folded into a 3-dimensional structure. The side chains of individual amino acid residues determine the interactions among amino acid residues, and ultimately the folding of the protein. Depending on the length and structural complexity, chains of amino acid residues are classified as oligopeptides, polypeptides, or proteins. An amino acid is a molecule that contains a carboxyl (–COOH) and an amino group (–NH2) attached to the same carbon atom, the ⍺-carbon. The identity of the amino acid is determined by its side chain or side residue, often called the R-group. The simplest amino acid is glycine, where the residue is a single hydrogen atom. Other amino acids carry more complex side chains. The side chain determines the chemical properties of the amino acid. For example, it may attract or repel water (hydrophilic or hydrophobic), carry a negative charge (acidic), or form hydrogen bonds (polar). Of all known amino acids, only 21 are used to create proteins in eukaryotes (the genetic code encodes only 20 of these). Amino acids are abbreviated using a three letter (e.g., Gly, Val, Pro) or one letter code (e.g., G, V, P). The linear chain of amino acid residues forms the backbone of the protein. The free amino group at one end is called the N-terminus, while t

 Core: Macromolecules

Density Gradient Ultracentrifugation

JoVE 5685

Density gradient ultracentrifugation is a common technique used to isolate and purify biomolecules and cell structures. This technique exploits the fact that, in suspension, particles that are more dense than the solvent will sediment, while those that are less dense will float. A high-speed ultracentrifuge is used to accelerate this process in order to separate biomolecules within a density…

 Biochemistry

Photometric Protein Determination

JoVE 5688

Measuring the concentration is a fundamental step of many biochemical assays. Photometric protein determination takes advantage of the fact that the more a sample contains light-absorbing substances, the less the light will transmit through it. Since the relationship between concentration and absorption is linear, this phenomenon can be used to measure the concentration in samples where it is…

 Biochemistry

DNA Isolation and Restriction Enzyme Analysis- Concept

JoVE 10628

The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our understanding of ourselves and other organisms. DNA isolation and profiling have been the fundamental first steps for many of the advancements in the past century; from identification of gene function, to revolutions of agriculture and…

 Lab Bio

Comparative Excretory Systems

JoVE 10998

Animals have evolved different strategies for excretion, the removal of waste from the body. Most waste must be dissolved in water to be excreted, so an animal’s excretory strategy directly affects its water balance.

Nitrogenous wastes are some of the most significant forms of animal waste. Nitrogen is released when proteins and nucleic acids are broken down for energy or conversion into carbohydrates and fats. Proteins are broken down into amino acids and nucleic acids into nitrogenous bases. The nitrogen-containing amino groups of amino acids and nitrogenous bases are then converted into nitrogenous wastes. Typical nitrogenous wastes released by animals include ammonia, urea, and uric acid. These excretory strategies involve tradeoffs between conserving energy and water. The various nitrogenous wastes reflect distinct habitats and evolutionary histories. For example, most aquatic animals are ammonotelic, meaning they directly excrete ammonia. This approach is less energy-intensive than converting ammonia into urea or uric acid before excretion, but also requires more water. For terrestrial organisms, which face perhaps no more significant regulatory threat than dehydration, water conservation is worth the extra energy cost. Ureotelic animals, like mammals and sharks, convert ammonia into urea before excretion. Urea is less toxic

 Core: Regulation and Excretion

Conditions on Early Earth

JoVE 11015

Around 4 billion years ago, oceans began to condense on earth while volcanic eruptions released nitrogen, carbon dioxide, methane, ammonia, and hydrogen into the primordial atmosphere. However, organisms with the characteristics of life were not initially present on earth. Scientists have used experimentation to determine how organisms evolved that could grow, reproduce, and maintain an internal environment. In the 1920s, the scientists Oparin and Haldane proposed the idea that simple biological compounds could have formed on the early earth. More than 30 years later, Stanley Miller and Harold Urey at the University of Chicago tested this hypothesis by simulating the conditions of the early earth's atmosphere and oceans in a laboratory apparatus. Using electricity as an energy source, the Miller-Urey experiment generated amino acids and other organic molecules, showing that the environment of early earth was conducive to the formation of biological molecules. More recent experiments have yielded comparable results and suggest that amino acids may have formed near areas of volcanic activity or hydrothermal vents in the ocean. Amino acids and small organic molecules may then have self-assembled to form more complex macromolecules. For instance, dripping amino acids or nucleotides into hot sand can result in the formation of the corresponding polymer

 Core: Evolutionary History

Levels of Organization

JoVE 10648

Biological organization is the classification of biological structures, ranging from atoms at the bottom of the hierarchy to the Earth’s biosphere. Each level of the hierarchy represents an increase in complexity that builds upon the previous level.

The most basic levels include atoms, molecules, and biomolecules. Atoms, the smallest unit of ordinary matter, are composed of a nucleus and electrons. Molecules comprise two or more atoms held together by chemical bonds, most commonly covalent, ionic, or metallic bonds. Biomolecules are molecules found in living organisms, including proteins, nucleic acids, lipids, and carbohydrates. Biomolecules are often polymers—large molecules that are created from smaller, repeating units. For instance, proteins are composed of amino acids, and nucleic acids are composed of nucleotides. Biomolecules can be endogenous or exogenous. Endogenous means that the biomolecule is produced inside a living organism. Biomolecules can also be consumed; for example, a cow gets carbohydrates from digesting grass (exogenous), but the grass must produce the carbohydrates through photosynthesis (endogenous). The next hierarchical level comprises subcellular structures called organelles. Organelles are made up of biomolecules and compartmentalize eukaryotic cells. Organelle means “little organ” as

 Core: Scientific Inquiry

Phosphodiester Linkages

JoVE 10685

Phosphodiester linkage is created when a phosphoric acid molecule (H3PO4) is linked with two hydroxyl groups (–OH) of two other molecules, forming two ester bonds and removing two water molecules. Phosphodiester linkage is commonly found in nucleic acids (DNA and RNA) and plays a critical role in their structure and function.

DNA and RNA are polynucleotides, or long chains of nucleotides, linked together. Nucleotides are composed of a nitrogen base (adenine, guanine, thymine, cytosine, or uracil), a pentose sugar and a phosphate molecule (PO 3−4). In a polynucleotide chain, nucleotides are linked together by phosphodiester bonds. A phosphodiester bond occurs when phosphate forms two ester bonds. The first ester bond already exists between the phosphate group and the sugar of a nucleotide. The second ester bond is formed by reacting to a hydroxyl group (–OH) in a second molecule. Each formation of an ester bond removes a water molecule. Inside the cell, a polynucleotide is built from free nucleotides that have three phosphate groups attached to the 5o carbon of their sugar. These nucleotides are thus called nucleotide triphosphates. During the formation of phosphodiester bonds, two phosphates are lost, leaving the nucleotide with one phosphate group that is attached to t

 Core: Macromolecules

RNA Analysis of Environmental Samples Using RT-PCR

JoVE 10104

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz


Reverse transcription-polymerase chain reaction (RT-PCR) involves the same process as conventional PCR — cycling temperature to amplify nucleic acids. However, while conventional PCR only amplifies…

 Environmental Microbiology

Dehydration Synthesis

JoVE 10681

Dehydration synthesis is the chemical process in which two molecules are covalently linked together with the release of a water molecule. Many physiologically important compounds are formed by dehydration synthesis, for example, complex carbohydrates, proteins, DNA, and RNA.

Sugar molecules can be covalently linked together by dehydration synthesis, also called condensation reaction. The resulting stable bond is called a glycosidic bond. To form the bond, a hydroxyl (-OH) group from one reactant and a hydrogen atom from the other form water, while the remaining oxygen links the two compounds. For each additional bond that is formed, another molecule of water is released, literally dehydrating the reactants. For example, individual glucose molecules (monomers) can undergo repeated dehydration synthesis to create a long chain or branched compound. Such a compound, with repeating identical or similar subunits, is called a polymer. Given the diverse set of sugar monomers, and variation in the location of the linkage, a virtually unlimited number of sugar polymers can be built. Plants produce simple carbohydrates from carbon dioxide and water in a process called photosynthesis. Plants store the resulting sugars (i.e., energy) as starch, a polysaccharide that is created from glucose molecules by dehydration synthesis. Cellulose is likewise buil

 Core: Macromolecules

Introduction to the Spectrophotometer

JoVE 5038

The spectrophotometer is a routinely used instrument in scientific research. Spectrophotometry is the quantitative measurement of how much a chemical substance absorbs light by passing a beam of light through the sample using a spectrophotometer.

In this video, basic concepts in spectrophotometry, including transmittance, absorbance and the Beer-Lambert Law …

 General Laboratory Techniques

Expression Profiling with Microarrays

JoVE 5547

Microarrays are important tools for profiling gene expression, and are based on complementary binding between probes that are attached to glass chips and nucleic acids derived from samples. Using these arrays, scientists can simultaneously evaluate the expression of thousands of genes. In addition, the expression profiles of different cells or tissue types can be compared, …

 Genetics

Contact-dependent Signaling

JoVE 10715

Contact-dependent signaling uses specialized cytoplasmic channels between cells that allow the flow of small molecules between them. In animal cells, these channels are called gap junctions. In plants, they are known as plasmodesmata.

Gap junctions form when two hemichannels, or connexons, join; one connexon from one cell coupling to a connexon of an adjacent cell. Each cell’s connexon is formed from six proteins creating a circular channel. There are over 20 different types of these proteins, or connexins, so there is substantial variation in how they come together as connexons and as gap junctions. Connexins have four transmembrane subunits with both their N- and C-terminus endings located intracellularly. The C-terminus has multiple phosphorylation sites so it can be activated by numerous different kinases- further adding to gap junction variety. Depending on the activating kinase, and the C-terminal amino acid residues of connexins that are phosphorylated, gap junctions can be partially or fully opened. This selectively allows small molecules to flow from one cell into another. A gap junction may also exclude by electrochemical charge. The selectivity of gap junctions allows a single cell to coordinate a complex multicellular response. However, some toxic molecules, matching the size and electrochemical preference of the gap junction, can also p

 Core: Cell Signaling

Cell Structure- Concept

JoVE 10587

Background

Cells represent the most basic biological units of all organisms, whether it be simple, single-celled organisms like bacteria, or large, multicellular organisms like elephants and giant redwood trees. In the mid 19th century, the Cell Theory was proposed to define a cell, which states:



Every living organism is made up of one or more cells.
The cells…

 Lab Bio

In ovo Electroporation of Chicken Embryos

JoVE 5156

Electroporation is a technique used in biomedical research that allows for the manipulation of gene expression via the delivery of foreign genetic material into cells. More specifically, in ovo electroporation is performed on early developing chicks (Gallus gallus domesticus) contained within their eggshells. In this procedure, DNA or knockdown constructs are first injected…

 Biology II

An Introduction to Transfection

JoVE 5068

Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of RNA into a cell is used to down-regulate the production of a specific protein by stopping translation. While the site of action for…

 Basic Methods in Cellular and Molecular Biology

An Overview of Gene Expression

JoVE 5546

Gene expression is the complex process where a cell uses its genetic information to make functional products. This process is regulated at multiple stages, and any misregulation could lead to diseases such as cancer.

This video highlights important historical discoveries relating to gene expression, including the…

 Genetics

Solid Phase Synthesis

JoVE 10349

Source: Vy M. Dong and Diane Le, Department of Chemistry, University of California, Irvine, CA


Merrifield's solid-phase synthesis is a Nobel Prize winning invention where a reactant molecule is bound on a solid support and undergoes successive chemical reactions to form a desired compound. When the molecules are bound to a solid…

 Organic Chemistry II

Histological Staining of Neural Tissue

JoVE 5206

In order to examine the cellular, structural and molecular layout of tissues and organs, researchers use a method known as histological staining. In this technique, a tissue of interest is preserved using chemical fixatives and sectioned, or cut into very thin slices. A variety of staining techniques are then applied to provide contrast to the visually uniform sections. In …

 Neuroscience

Quantifying Environmental Microorganisms and Viruses Using qPCR

JoVE 10186

Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Bradley Schmitz


Quantitative polymerase chain reaction (qPCR), also known as real-time PCR, is a widely-used molecular technique for enumerating microorganisms in the environment. Prior to this approach, quantifying microorganisms…

 Environmental Microbiology

Live Cell Imaging of Mitosis

JoVE 5642

Mitosis is a form of cell division in which a cell’s genetic material is divided equally between two daughter cells. Mitosis can be broken down into six phases, during each of which the cell’s components, such as its chromosomes, show visually distinct characteristics. Advances in fluorescence live cell imaging have allowed scientists to study this process in…

 Cell Biology

Cell Cycle Analysis

JoVE 5641

Cell cycle refers to the set of events through which a cell grows, replicates its genome, and ultimately divides into two daughter cells through the process of mitosis. Because the amount of DNA in a cell shows characteristic changes throughout the cycle, techniques known as cell cycle analysis can be used to separate a population of cells according to the different phases …

 Cell Biology

Macromolecules - Prep Student

JoVE 10589

Solution Preparation
CAUTION: It is important to always use care when handling reagents. For example, iodine will stain clothes and skin so wear gloves, goggles, and a lab coat when using this reagent.
To prepare a 1 M glucose solution, begin by using a stir bar to dissolve 180.2 g of glucose in 500 mL of distilled water, increasing the volume to…

 Lab Bio

pre-mRNA processing

JoVE 11003

In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA, to distinguish it from mature mRNA.

Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it. This 5’ cap helps the cell distinguish mRNA from other types of RNA in the cell and plays a role in subsequent translation. During or shortly after transcription, a large complex called the spliceosome cuts out various parts of the pre-mRNA transcript, rejoining the remaining sequences. RNA sequences that remain in the transcript are called “exons” (expressed sequences) while portions removed are called “introns”. Interestingly, a single RNA segment can be an exon in one cell type and an intron in another. Similarly, a single cell can contain multiple variants of a gene transcript that has been alternatively spliced, enabling the production of multiple proteins from a single gene. When transcription is completed, an enzyme adds approximately 30-200 adenine nucleotides to the 3’ end of the pre-mRNA molecule. This poly-A tail protects the mRNA fr

 Core: Gene Expression

What Are Osmoregulation and Excretion?

JoVE 11001

Organisms must keep bodily fluids at a constant temperature and pH while maintaining specific solute concentrations in order to support life functions. Osmoregulation is the process that balances solute and water levels.

Osmosis is the tendency of water to move from solutions with lower ion concentrations, or osmolarities, to those with higher ion concentrations. Osmosis occurs in response to differences in the molecular concentrations of solutions separated by a semipermeable membrane. Bodily fluids, which are separated by such membranes, contain water, non-electrolytes, and electrolytes—solutes that dissolve into ions in water. Both electrolytes and non-electrolytes influence osmotic balance. However, since the more important factor to osmosis is solute number, rather than size, the contribution of electrolytes is more significant. Unlike water, electrolytes cannot diffuse passively through membranes but rely on facilitated diffusion and active transport. In facilitated diffusion, protein-based channels move solutes across membranes. Conversely, energy is used to move ions against concentration gradients in active transport. When animals ingest food, material that cannot be used is excreted from the body. Excretory systems in nature involve tradeoffs between conserving energy and water. Nitrogen is among the most significant

 Core: Regulation and Excretion

Cofactors and Coenzymes

JoVE 10975

Enzymes require additional components for proper function. There are two such classes of molecules: cofactors and coenzymes. Cofactors are metallic ions and coenzymes are non-protein organic molecules. Both of these types of helper molecule can be tightly bound to the enzyme or bound only when the substrate binds.

Cofactors are present in ~30% of mature proteins. They are frequently incorporated into an enzyme as it is folded and are involved in the enzyme’s catalytic activity. Magnesium is an essential cofactor for over 300 enzymes in the human body, including DNA polymerase. In this case, the magnesium ion aids in the formation of the phosphodiester bond on the DNA backbone. Iron, copper, cobalt, and manganese are other common cofactors. Many vitamins are coenzymes, as they are nonprotein, organic helper molecules for enzymes. For example, biotin—a type of B vitamin—is important in a variety of enzymes that transfer carbon dioxide from one molecule to another.  Biotin, vitamin A and other vitamins must be ingested in our diet, as they cannot be made by human cells.

 Core: Metabolism

DNA Isolation

JoVE 10814

DNA from cells is required for many biotechnology and research applications, such as molecular cloning. To remove and purify DNA from cells, researchers use various methods of DNA extraction. While the specifics of different protocols may vary, some general concepts underlie the process of DNA extraction.

First, cells need to be lysed—broken open—to release the DNA into solution. Cells can be physically lysed using equipment such as a homogenizer, sonicator, or bead beater, which grind or otherwise apply force to break the cells open. Often, substances such as detergent are added during lysis to chemically disrupt the lipid-based cell membranes—helping release the DNA from the nucleus and cell. The spinning in a centrifuge sediments the cell debris to the bottom, and the lysate—containing cellular materials—is collected for further processing. The DNA must then be separated from other cellular molecules, such as RNA and proteins. Therefore, enzymes such as RNase and Proteinase K are often added during or after lysis to degrade RNA and proteins, respectively. Additionally, organic solvents such as phenol and chloroform are commonly used to separate DNA from protein. Typically, the sample is vortexed with phenol-chloroform and then centrifuged to separate the aqueous and organic phases in the tube. The DNA-containing aqueous p

 Core: Biotechnology

Taxonomy

JoVE 10653

Taxonomy is the science of defining and naming groups of biological organisms based on shared characteristics. It uses a hierarchy of increasingly inclusive categories with Latin names. The smallest units of taxonomy, species and genus, are used to assign a formal, taxonomic name to each species in a system. This classification system, referred to as binomial nomenclature, was formalized by Carolus Linnaeus in the 18th century. The hierarchy that Carolus Linnaeus first proposed is still used today, although it has been expanded upon. The order of ranking—from the highest or largest group to the smallest or most specific—is as follows: domain, kingdom, phylum, class, order, family, genus, and species. Beginning from the smallest unit of taxonomy, similar species are grouped into the same genus. For example, the arctic hare and the black-tailed jackrabbit both belong to the genus Lepus; however, they belong to different species—arcticus and californicus, respectively. Within an organism’s taxonomic name, both the genus and species are italicized, and the first letter of the genus is capitalized. This two-part format for naming and categorizing specific organisms is referred to as binomial nomenclature. Members of the same genus belong to the same family. For example, hares and rabbits belong t

 Core: Scientific Inquiry

DNA Isolation and Restriction Enzyme Analysis - Prep Student

JoVE 10575

Preparation of Solutions and Materials
NOTE: For this lab to run smoothly, it is recommended to not exceed 12 samples per laboratory class.
First gather the necessary materials, including popsicle sticks, funnels, 15 mL conical tubes, micropipettes, ice buckets with ice, small cups and microcentrifuge tubes.
Next set the water bath used in…

 Lab Bio

X-ray Diffraction

JoVE 10446

Source: Faisal Alamgir, School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA


X-ray diffraction (XRD) is a technique used in materials science for determining the atomic and molecular structure of a material. This is done by irradiating a sample of the material with incident X-rays and then measuring the …

 Materials Engineering

Testing For Genetically Modified Foods

JoVE 10044

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University


Genetic modification of foods has been a controversial issue due to debated concerns over health and environmental safety. This experiment demonstrates technical understanding of how food DNA is genetically identified, allowing for educated decision making about …

 Environmental Science

Mouse Genotyping

JoVE 5160

Even though the human genome was mapped over 10 years ago, scientists are still far from understanding the function of every human gene! One way to evaluate how a gene functions is to disrupt the sequence encoding it and then evaluate the impact of this change (the phenotype) on the animal’s biology. This approach is commonly used in the mouse (Mus musculus), since it shares a…

 Biology II

Basic Chick Care and Maintenance

JoVE 5154

Chicks (Gallus gallus domesticus) are a valuable research tool, not only for studying important concepts in vertebrate development, neuroscience, and tumor biology, but also as an efficient system in which to propagate viruses. Although eggs can be purchased from external suppliers and working with chicks requires very little specialized equipment, an understanding of proper handling…

 Biology II

Regulating Temperature in the Lab: Preserving Samples Using Cold

JoVE 5042

Preservation of laboratory samples, specimens, and reagents using extreme cold is routinely performed in biomedical research labs. This video will discuss some of the methods for keeping laboratory samples cold and will explain the correct cooling method to use for each experimental requirement.


For example, cooling agents, such as ice…

 General Laboratory Techniques
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