<h5 id="121-microscopy">12.1 Microscopy</h5> <ul> <li>Microscopy is essential in biological studies, allowing the visualization of structures beyond the scope of the human eye.</li> <li>The first microscope, designed by Robert Hooke in 1665, was a simple microscope using magnifying lenses. Hooke observed the slices of cork and coined the term “Cellulae or cell”.</li> <li>The cell theory was proposed in 1838 by Matthias Jacob Schleiden and Theodor Schwann, based on their observations of cells in plants and animals.</li> <li>Modern microscopy allows for highly magnified, three-dimensional images of minute structures, and even the visualization of DNA molecules of bacteria and viruses using electron microscopic techniques.</li> <li>The link provided contains an image of a microscope (Fig. 12.1) but it can’t be displayed in this text-based interface.</li> </ul> <p>======</p> <h5 id="1211-magnification-and-resolution">12.1.1 Magnification and Resolution</h5> <ul> <li>Magnification of a microscope is the ability to increase the retinal image size. It is measured as the ratio of the size of the retinal image formed with the help of the microscope to the size of the retinal image formed without using the microscope.</li> <li>The magnification (M) of a lens is calculated using the formula M = f/(f-d), where f is the focal length of the lens and d is the distance of the object from the lens.</li> <li>A microscope used in a laboratory is typically a compound microscope, which consists of two sets of lenses: an objective lens and an eyepiece.</li> <li>The magnification of a microscope is the product of the magnifying power of the objective lens and the magnifying power of the eyepiece (M = M_o * M_e).</li> <li>Resolving power is the ability of a microscope to form separate images of two objects located very close to each other. It can be measured by the smallest distance between two points.</li> </ul> <p>======</p> <h5 id="1212-functioning-of-a-light-microscope">12.1.2 Functioning of a light microscope</h5> <ul> <li>A compound microscope consists of a base, stage, nose piece, objective lenses, eye piece, adjustment screws, light source, and condenser.</li> <li>The objective lenses and eye pieces have different magnifying powers, typically in a student microscope, the eyepiece has a magnifying power of $10 oxtimes$ or $15 oxtimes$, and the objective lenses have magnifying power of $4 oxtimes$, $10 oxtimes$, $40 oxtimes$ or $45 oxtimes$, and $100 oxtimes$.</li> <li>The distance between the objective lens and the object on the stage can be adjusted using coarse and fine adjustment screws.</li> <li>The light source illuminates the object, and the condenser focuses light on the object, facilitating the formation of an image through the objective lens and eye piece.</li> <li>Staining the object with specific dyes or stains, such as Carmine, Eosin, Safranin, Methylene blue, and Giemsa, is necessary to distinguish different regions of the object in bright field microscopy.</li> </ul> <p>======</p> <h5 id="1213-different-forms-of-microscopy">12.1.3 Different forms of microscopy</h5> <ul> <li>Dark Field Microscopy is a form of microscopy where light falling on the object from the central condenser is blocked, and illumination of the object is done by an oblique light beam. This is used to easily detect mitochondria, nuclei, vacuoles, etc.</li> <li>Phase Contrast Microscopy is a form of microscopy where the wave amplitude and phase of light passing through the transparent object is changed, resulting in a varied contrast of different regions of the object. This is especially helpful in the study of cell organelles and chromosomes.</li> <li>Fluorescence Microscopy is a form of microscopy where the object to be seen is stained with a fluorophore to study a specific part of an organelle or molecule. This is helpful in identifying bacteria or viruses to know the cause of infection and immunodiagnosis.</li> <li>Transmission Electron Microscopy is a type of electron microscopy where the ultra-thin heavy metal salt (of lead, tungsten, etc.) coated section of the object or specimen is placed in such a way that the electron beam passes through it to create the image.</li> <li>Scanning Electron Microscopy is a type of electron microscopy where reflected electron beam from the gold or platinum coated surface of the object creates the image. This is used to generate a highly magnified and resolved image of the object surface.</li> <li>Confocal Microscopy is a sophisticated microscopic imaging technique used to resolve detailed structures within fixed cells/tissues and gives sharp images of the objects. This involves fluorescently labeling the object and then analyzing it under a confocal microscope in high resolution.</li> </ul> <p>======</p> <h5 id="122-centrifugation">12.2 Centrifugation</h5> <ul> <li>Centrifugation is a technique used for separating particles or molecules based on their densities through the use of a centrifuge.</li> <li>A centrifuge consists of a base, a rotating container (spinning vessel/rotor), and a lid, with the spinning vessel containing several centrifuge tubes.</li> <li>The cell extract or mixture is placed in centrifuge tubes and spun at a specific speed (rpm) for a certain period of time, leading to the settling of particulate material at the bottom of the tubes.</li> <li>The particles or molecules are separated based on their densities under the influence of gravitational force (g) and centrifugal force.</li> <li>Different types of centrifuges are used depending on their specific purpose.</li> </ul> <p>======</p> <h5 id="1221-basic-principles-of-sedimentation">12.2.1 Basic principles of Sedimentation</h5> <ul> <li>Sedimentation is the process of particles in suspension settling out of a fluid due to gravity or centrifugal forces.</li> <li>The motion of these particles through the fluid is influenced by the forces acting on them.</li> <li>Gravity and centrifugal forces are the main forces responsible for sedimentation.</li> <li>Particles settle out of the fluid and come to rest against a barrier during sedimentation.</li> <li>This process is driven by the particles’ motion in response to the forces they encounter in the fluid.</li> </ul> <p>======</p> <h5 id="1222-types-of-centrifuges">12.2.2 Types of Centrifuges</h5> <ul> <li>There are several types of centrifuges used for research purposes: table top/clinical centrifuge or microfuge, high-speed centrifuge, ultracentrifuge, and differential centrifuge.</li> <li>Centrifugation types include differential centrifugation, density-gradient centrifugation, and ultracentrifugation.</li> <li>Differential centrifugation separates particles of different size and density based on their sedimentation rate.</li> <li>Density-gradient centrifugation separates biological particles of similar size but differing in densities by developing a density gradient in centrifuge tubes.</li> <li>Ultracentrifugation, carried out at very high speed (100,000 x g or more), separates molecules and requires light passage through the biological particles for proper concentration distribution measurement.</li> </ul> <p>======</p> <h5 id="123-electrophoresis">12.3 Electrophoresis</h5> <ul> <li>Electrophoresis is a method of separation based on the charge to mass ratio of macromolecules in an electric field.</li> <li>It was first observed in 1807 by Russian professors Peter Ivanovich Strakhov and Ferdinand Frederic Reuss.</li> <li>The principle of electrophoresis relies on the migration of electrically charged biological molecules such as nucleotides, DNA, RNA, peptides, and proteins towards the cathode or anode.</li> <li>The mobility of a molecule is influenced by its size and charge, allowing for separation.</li> <li>At a given pH, these particles exist in solution as electrically charged species, either as cations or anions.</li> </ul> <p>======</p> <h5 id="1231-agarose-gel-electrophoresis">12.3.1 Agarose Gel Electrophoresis</h5> <ul> <li>Agarose gel electrophoresis uses a gel matrix made of agarose molecules for DNA separation.</li> <li>The gel, a slab of jelly-like material, is placed in a gel box with a salt-containing buffer that can conduct current.</li> <li>DNA molecules, being negatively charged, migrate towards the positive electrode; shorter DNA fragments move faster, allowing size-based separation.</li> <li>The voltage range for running an agarose DNA gel is 80-120 V.</li> <li>This process helps determine the absolute size of different DNA fragments in a sample.</li> </ul> <p>======</p> <h5 id="1232-visualising-the-dna-fragments">12.3.2 Visualising the DNA fragments</h5> <ul> <li>The equipment used for visualization of target DNA is an ultra-violet (UV) trans-illuminator.</li> <li>Ethidium bromide (EtBr) is a well-known stain used for visualizing DNA, which can be mixed in the gel mixture, electrophoresis buffer, or stained in the gel after it is run.</li> <li>Molecules of EtBr intercalate within DNA bases and fluoresce under UV light, making it easier to visualize DNA bands.</li> <li>Despite its advantages, ethidium bromide is a potential carcinogen, so it must be handled with care.</li> <li>Figure 12.5 shows the visualization of DNA bands under UV light.</li> </ul> <p>======</p> <h5 id="1233-polyacrylamide-gel-electrophoresis-page">12.3.3 Polyacrylamide Gel Electrophoresis (PAGE)</h5> <ul> <li>PAGE is a method to separate proteins in a mixture based on their size.</li> <li>Anionic detergent SDS is used to give proteins negative charge for uniform charge.</li> <li>Proteins are separated in a gel matrix of polyacrylamide produced by polymerisation of acrylamide and BIS.</li> <li>The degree of polymerisation or cross-linking of polyacrylamide can be controlled by adjusting the concentration of acrylamide and BIS.</li> <li>Hardness of the gel affects the resolution of separation, with loose gels allowing higher molecular weight molecules to move faster and hard gels restricting larger molecules.</li> </ul> <p>======</p> <h5 id="1234-tracking-dye">12.3.4 Tracking dye</h5> <ul> <li> <p>Tracking dyes are anionic and included in the sample buffer to follow the progress of DNA, RNA, and proteins during electrophoresis.</p> </li> <li> <p>Common tracking dyes include bromophenol blue, xylene cyanol, and Orange G, each with varying electrophoretic mobility.</p> </li> <li> <p>Bromophenol blue is a small negatively charged molecule that moves ahead of most proteins and nucleic acids, signaling the end of electrophoresis.</p> </li> <li> <p>Coomassie Brilliant Blue R-250 is a popular protein stain that specifically binds to proteins, making them visible as blue bands on a clear background.</p> </li> <li> <p>Agar, a jelly-like substance, was discovered as a solidifying agent for bacterial culture by Fanny Eilshemius Hesse, and its primary component, agarose, is used in molecular biology for gel electrophoresis.</p> </li> </ul> <p>======</p> <h5 id="124-enzyme-linked-immunosorbent-assay-elisa">12.4 Enzyme-Linked Immunosorbent Assay (ELISA)</h5> <ul> <li>ELISA is a quantitative method for measuring antigen and antibody concentration, invented by Eva Engvall and Peter Perlman in 197</li> </ul> <p>======</p> <h5 id="125-chromatography">12.5 Chromatography</h5> <ul> <li> <p>Chromatography is a separation procedure involving a mobile phase and a stationary phase, where solutes in a mixture move at rates proportional to their relative affinity for the stationary phase.</p> </li> <li> <p>Different types of chromatography include:</p> <ul> <li>Adsorption Chromatography (ADC): Based on adsorption of solute molecules onto polar adsorbents, using solvent properties to aid mobile-phase selection.</li> <li>Liquid-liquid Partition Chromatography (LLC): Relying on partition coefficients of solute molecules between two immiscible solvents.</li> <li>Ion-exchange Chromatography (IEC): Using adsorption of solute ions on ion exchange resin particles by electrostatic forces, often used for fractionation of antibiotics and proteins.</li> <li>Gel Filtration Chromatography: Based on the size of molecules to be separated, using gel particles of distinct porosity.</li> <li>Affinity Chromatography (AFC): Exploiting the binding specificity of biomolecules, using a binding molecule called a ligand to retain solutes with appreciable affinity.</li> <li>High-pressure/performance Liquid Chromatography (HPLC): Based on general principles of chromatography, using small stationary phase particles and high pressure for the mobile phase.</li> </ul> </li> <li> <p>Chromatography is used in laboratories for sample analysis and as a preparative technique for large-scale purification of biomolecules, suitable for recovery of high-purity therapeutics and pharmaceuticals.</p> </li> <li> <p>Different chromatographic methods are available for purification of proteins, peptides, amino acids, nucleic acids, alkaloids, vitamins, steroids, and many other biological materials.</p> </li> </ul> <p>======</p> <h5 id="126-spectroscopy">12.6 Spectroscopy</h5> <ul> <li>UV/Visiblespectroscopy is used for the detection of functional groups, extent of conjugation, and determines the configurations of geometrical isomers. Atomic absorptionspectroscopy in UV/Visible range determines the amount of various levels of metals and other electrolytes within samples.</li> <li>Infrared spectroscopy is used for determining the functional groups. Ramanspectroscopy is used for contaminant identification, gemstone and mineral identification.</li> <li>Nuclear magneticresonancespectroscopy, which uses radio waves, provides information about the structure and chemical environment of atoms.</li> <li>X-ray absorptionspectroscopy determines the elemental composition and chemical bonding of molecules. Emission spectroscopy, such as atomic emissionspectroscopy, is used for detection of trace metals, minerals, sodium, potassium and lithium.</li> <li>Mass spectrometer is used for analysis of proteins, peptides, checking waterquality and food contamination. Photoluminescence, such as fluorescencespectroscopy, is used for detection of many of organic compounds, numerous aromatic active substances in drug. Phosphorescencespectroscopy is not mentioned with any specific use in the text.</li> </ul> <p>======</p> <h5 id="1261-colorimetry">12.6.1 Colorimetry</h5> <ul> <li>Colorimetry is a technique that measures the interaction of light energy with colored solutions.</li> <li>It uses a colorimeter instrument to measure transmittance and absorbance of light passing through a liquid sample.</li> <li>The intensity of color is directly proportional to the concentration of the compound being measured, based on Beer-Lambert’s law.</li> <li>The three main components of a colorimeter are a tungsten-filament lamp, filter, cuvette containing sample, and a photocell for detecting transmitted light.</li> <li>Colorimeters are inexpensive, easily transportable, and used for quantitative analysis of colored compounds.</li> </ul> <p>======</p> <h5 id="1262-uv-visible-spectrophotometry">12.6.2 UV-visible Spectrophotometry</h5> <ul> <li>UV-visible spectrophotometry measures the amount of light absorbed at each wavelength in the UV and visible regions of the electromagnetic spectrum.</li> <li>A spectrophotometer is a more advanced type of colorimeter, using a prism or grating to provide monochromatic light, as opposed to the broad range of wavelengths allowed by filters in a colorimeter.</li> <li>The term “photometer” refers to a device for measuring light, and “spectro” indicates the whole range of continuous wavelengths that the light source is capable of producing.</li> <li>The main components of a spectrophotometer include light sources (UV and visible), a wavelength selector (monochromator), sample containers (cuvette), a detector, and a signal processor.</li> <li>Fig. 12.13 provides a visual representation of a UV-visible spectrophotometer, which consists of these components.</li> </ul> <p>======</p> <h5 id="127-mass-spectrometry">12.7 Mass Spectrometry</h5> <ul> <li>Mass spectrometry is a technique used for identification, quantification, and analysis of molecular structure and properties.</li> <li>The process involves ionization of the sample, usually by electron ionization, which can be with or without fragmentation.</li> <li>Ions are then characterized by their mass-to-charge ratios (m/z) and relative abundances.</li> <li>The unit of measurement of mass is Dalton (Da), where 1 Da is equal to 1/12th of the mass of a single atom of the isotope of carbon-12.</li> <li>The major components of mass spectrometry are: <ul> <li>Ion source: produces gaseous ions from the substance being studied.</li> <li>Analyzer: resolves ions into their characteristic mass components according to their mass-to-charge ratio.</li> <li>Detector system: detects ions and records the relative abundance of each resolved ionic species.</li> </ul> </li> </ul> <p>======</p> <h5 id="128-fluorescence-in-situ-hybridisation-fish">12.8 Fluorescence In Situ Hybridisation (FISH)</h5> <ul> <li>FISH is a cytogenetic technique that uses fluorescent molecules to bind to specific regions of chromosomes, helping identify the location of particular genes.</li> <li>The process involves five steps: designing a complementary probe, placing chromosomes on a microscope slide and denaturing them, denaturing the probe and adding it to the slide, washing off excess probe, and observing the chromosome under a fluorescent microscope.</li> <li>The main components of FISH are a fluorescent DNA molecule (probe) and the chromosome (target sequence).</li> <li>FISH is useful in understanding chromosomal abnormalities and is an important tool in the field of genetics.</li> <li>The probe, when hybridized, will show as one or more fluorescent signals in the microscope, depending on the number of sites it can bind to.</li> </ul> <p>======</p> <h5 id="chromosome-painting">Chromosome Painting</h5> <ul> <li>Chromosome painting is a technique that uses a special probe, called Multifluor FISH probe, to generate a karyotype with each chromosome appearing in a different color.</li> <li>The process involves preparing DNA sequences for each chromosome and labeling them with unique combinations of fluorochromes to produce a distinct color.</li> <li>The fluorescent DNA probes and metaphase chromosomes are then mixed and visualized under a fluorescent microscope.</li> <li>Osamu Shimomura, a Japanese organic chemist and marine biologist, discovered Green Fluorescent Protein (GFP) accidentally from jellyfish in 1962.</li> <li>Shimomura, along with Martin Chalfie and Roger Tsien, received the Nobel Prize in Chemistry in 2008 for the discovery and development of GFP.</li> </ul> <p>======</p> <h5 id="129-dna-sequencing">12.9 DNA Sequencing</h5> <ul> <li>DNA sequencing is the process of determining the order of nucleotides (A, T, G, C) in a DNA molecule.</li> <li>The sequence of bases in DNA is crucial as it contains the heritable genetic information that influences health, disease susceptibility, and reactions to medications.</li> <li>Understanding a specific DNA sequence can help explain genetic conditions and potentially lead to treatment development.</li> <li>Alterations in DNA sequences can result in altered or non-functional proteins, which can have harmful effects.</li> <li>DNA sequencing technology is essential for studying the structure, function, and evolutionary history of cloned DNA and has applications in various fields such as basic research, diagnostic, forensic, environmental, agricultural, and more.</li> <li>Modern DNA sequencing technology has enabled the rapid sequencing of complete DNA sequences of various organisms, including the human genome.</li> </ul> <p>======</p> <h5 id="1291-dna-sequencing-methods">12.9.1 DNA sequencing methods</h5> <ul> <li>DNA sequencing methods have historically been categorized into two main methods: Enzymatic method (Sanger’s method) and Chemical degradation method (Maxam and Gilbert method).</li> <li>In Sanger’s method, dideoxynucleotides (lacking the normal-OH group at 3’ position) are incorporated into the newly synthesized chain, leading to termination of synthesis of the new strand. This method is considered the gold standard for DNA sequencing.</li> <li>The template DNA is mixed with a primer, four normal dNTPs (one of which is labelled radioactively or fluorescently), and a different ddNTP in each of the four tubes. Following DNA polymerase addition, synthesis of new strands of DNA complementary to the template begins.</li> <li>In the Chemical degradation method (Maxam and Gilbert method), the DNA fragment is cleaved in a base-specific manner using chemicals. The DNA is labelled at the 5’ end using enzyme polynucleotidyl kinase and gamma-32P labelled nucleotide. The fragments generated are subsequently separated using high resolution PAGE to resolve the sequence order.</li> <li>The fragments are ordered by size, and the location of radiolabeled DNA fragments is shown as a series of dark bands on X-ray film. The sequence of the DNA molecule can be deduced from the location of these bands.</li> </ul> <p>======</p> <h5 id="1292-automated-dna-sequencing">12.9.2 Automated DNA sequencing</h5> <ul> <li>Automated DNA sequencing is primarily based on the Sanger method, using fluorescent labeled dideoxynucleotides.</li> <li>The slab gel is replaced by polymer filled capillary tubes, allowing for faster and more reliable sequencing.</li> <li>In a single tube, four dideoxynucleotides are labeled with different fluorescent dyes, enabling all reactions to be carried out simultaneously.</li> <li>A fluorimeter and computer detect and record the fluorescent dye attached to the fragments, determining the sequence by the order of the dyes coming off the gel.</li> <li>Next-generation Sequencing (NGS) methods, such as Roche/454 FLX sequencing, Solexa/Illumina, and SOLiD platforms, facilitate sequencing of very large DNA, enabling massively parallel sequencing reactions.</li> </ul> <p>======</p> <h5 id="1210-dna-microarray">12.10 DNA Microarray</h5> <ul> <li>DNA microarray technology is a high-throughput hybridization-based method used for the analysis of a large number of DNA fragments in parallel, primarily for gene expression quantification.</li> <li>The principle of DNA microarray technology is based on the pairing of two DNA strands through hydrogen bonds between complementary nucleotide bases. It utilizes complementary DNA sequences to hybridize to immobilized DNA fragments on the chip.</li> <li>Thousands of single-stranded DNA (ssDNA) segments, corresponding to gene transcripts (mRNA) or other genomic regions, are immobilized on a small solid surface, referred to as microarray chips. These chips are usually made of glass or nylon and coated with a special surface coating for DNA spotting or in situ synthesis of oligonucleotides.</li> <li>A typical microarray experiment includes mRNA extraction, probe labelling, hybridization and washing, and scanning and data analysis. mRNA is converted into complementary DNA (cDNA) for stability and represents actively transcribing genes in the cell.</li> <li>Labelled DNA fragments are hybridized with the microarray chip, and the number of duplexes formed reflects the number of DNA segments complementary to its probe. Microarray data analysis software uses different colors to represent the expression level of genes in one condition with respect to another. Genes whose expression increases or decreases are referred to as upregulated or downregulated genes, respectively.</li> </ul> <p>======</p> <h5 id="1211-flow-cytometry">12.11 Flow Cytometry</h5> <ul> <li>The technique to quantify the expression of a large number of genes is Microarray (option b).</li> <li>SEM and TEM (option a) differ in that SEM images the surface of a sample by scanning it with a focused beam of electrons, while TEM passes the electrons through the sample to form an image.</li> <li>In agarose gel electrophoresis (option d), the principle is based on the movement of negatively charged molecules (like DNA and proteins) towards the positively charged electrode when an electric field is applied.</li> <li>Ethidium bromide is a tracking dye used to track DNA and proteins during electrophoresis. If forgotten, it would be harder to visualize and determine the sizes of the molecules.</li> <li>Gel B is harder (option a) and offers greater friction to proteins (option b). It would be used to separate a mixture containing both low and high molecular weight proteins (option d).</li> <li>A chromatogram is a graph produced by chromatography to visualize the separation of different components of a mixture.</li> <li>FISH (option d) is a technique used to locate specific DNA sequences in chromosomes. It’s applied in chromosome painting by labeling probes with fluorescent dyes to identify specific chromosomes or chromosome regions.</li> <li>Spectroscopy techniques (option e) have various applications, including determining the structure and properties of molecules, identifying and quantifying substances, and monitoring chemical reactions.</li> <li>A UV-visible spectrophotometer (option f) consists of a light source, a monochromator, a sample holder, and a detector. It measures the intensity of light absorbed or transmitted by a sample to determine its concentration or properties.</li> <li>Sanger’s method (option g) uses DNA polymerase to add dideoxynucleotides (ddNTPs) to a DNA strand, terminating extension at specific bases. Maxam-Gilbert sequencing (option h) involves radioactive labeling and chemical cleavage of DNA at specific bases.</li> <li>Flow cytometry (option i) measures physical and chemical characteristics of cells or particles by suspending them in a fluid and passing them through a laser or other light source to detect and analyze the scattered or emitted light.</li> </ul> <p>======</p>