Biotechniques (BMS 34010A) Fall 2023-2024 PDF

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Basic principles: Biosafety in the laboratory What is Lab Safety?  The laboratory setting presents several hazards that must be considered when completing any lab exercise.  Laboratory safet...

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Basic principles: Biosafety in the laboratory What is Lab Safety?  The laboratory setting presents several hazards that must be considered when completing any lab exercise.  Laboratory safety includes procedures that are designed for the protection of all laboratory personnel and the safe use of laboratory equipment.  It also involves strict protocols on the disposal of contaminated materials and chemicals.  Potential Laboratory Hazards  The science laboratory can present a number of hazards to laboratory workers.  These may include corrosive chemicals, sharp tools/glass items, and the use of open flames.  Extreme caution should be used when working with open flames, such as those on a Bunsen burner. Always know when the flame is turned on and pay attention to what materials you have near the flame  Potential Laboratory Hazards  Infectious organisms may also pose a threat to laboratory workers.  In fact, microorganisms are classified into four biosafety levels based on their ease of transmission and pathogenicity. Safety standards in the lab have been developed for working with each biosafety level.  Potential Laboratory Hazards  Most undergraduate laboratories only use BSL 1 organisms, well known organisms that do not usually cause disease in immuno compentent persons.  Working with BSL 4 organisms requires extensive safety measures, such as the personal protective gear shown in this image. These organisms, including Marburg virus and hantavirus, are handled only in specialized laboratories at places like the Centers for Disease Control.  Standard Practices in the Laboratory  Lab coats are an important part of safety when conducting any lab exercise. These can protect a you from contamination by microorganisms or corrosive substances. ▪ They should be long enough in both the sleeves and body to provide good coverage, but not so long that the coat could accidentally get caught on something or be dragged through a hazardous substance. ▪ They should not be so tight that they restrict movements.  REMEMBER: Lab coats are not chemical protection suits! DO immediately remove a lab coat if on fire or there is obvious hazardous contamination.  Standard Practices in the Laboratory  Gloves are also commonly worn to protect the hands from contamination or contact with chemicals/staining reagents.  Not all experiments require the use of gloves, as there may be advantages or disadvantages to their use in the lab.  REMEMBER: Gloves should never touch door handles, elevator buttons, telephones, card swipes, or any surfaces outside of the laboratory. ▪ If you transport materials from labs through common areas, use an ungloved hand to touch common surfaces and a gloved hand to carry the items.  Standard Practices in the Laboratory  Now that you are aware of some potential hazards in the laboratory, let's review some standard safety practices that should always be followed.  First of all, you should never put anything in your mouth while in the laboratory. This includes consuming food, drinking, and chewing gum. You also should never attempt to pipette substances by mouth. This strict rule should be followed to avoid the intake of dangerous substances.  Secondly, you should not apply makeup or adjust contacts while in the laboratory.  Standard Practices in the Laboratory  When conducting lab experiments, the lab bench should be kept clean. You should only have the appropriate lab materials, the lab manual, and a pencil or pen on the lab bench.  Cell phones, purses, book bags, and other personal items should always be safely stowed away from the benchtop.  Do not leave active experiments unattended. Never leave anything that is being heated or is visibly reacting unattended.  Standard Practices in the Laboratory  Before and after every lab experiment, it is good practice to disinfect the benchtop.  This prevents contamination during the exercise and leaves the lab environment clean for the next students.  Standard Practices in the Laboratory  Keep your hands clean. Later in this module, you will also review correct hand washing technique.  Never run in the laboratory.  Do not sit on laboratory benches.  The performance of unauthorized experiments is strictly forbidden. Never work in the laboratory without the supervision.  Standard Practices in the Laboratory  Whenever working with open flames, it is important to tie back long hair and secure loose clothing SAY NO TO: long hair long nails loose clothing Standard Practices in the Laboratory  Goggles, safety glasses or face shields are required when working with UV light and any procedure where aerosols or chemicals may splash into the eyes.  If you wear glasses, safety glasses must be worn over them.  Standard Practices in the Laboratory  During laboratory work, there is a great risk of spilling chemicals on the floor or dropping sharp objects.  Always wear closed toed shoes to avoid contact between chemical substances and the bare skin during spilling accidents. Closed toed shoes may also prevent cuts to the feet in the case of dropping a sharp tool like a dissection scalpel.  Laboratory Safety Equipment  It is also important to know the location of safety equipment in the laboratory classroom.  These include the eyewash and chemical shower used in the event of skin or eye contamination.  A first aid kit and fire extinguisher should also be located somewhere in the laboratory classroom.  Laboratory Safety Equipment  Many institutions have developed chemical hygiene plans, written policies that protect lab personnel from health hazards associated with the use of chemicals in the lab.  Therefore you should always make use of fume hoods or biosafety cabinets as directed by your instructor or the lab exercise procedure.  Lab Emergencies  In the event of a lab emergency, you should immediately notify your instructor. Report any spills, accidents, or injuries.  This includes chemical and biological spills, personal injuries, broken glass, broken laboratory equipment, and other obvious emergencies.  Your instructor will guide you in appropriately dealing with these issues.  Biohazard Bin  Biohazardous waste must be placed into appropriately labeled bins or bags.  This includes non reusable items that have been in contact with infectious organisms or body fluids and animal tissue that is ready to be discarded. Any gloves worn during an exercise involving biohazardous materials should also be placed in this bin.  Items placed in the biohazard bag may be sterilized using an autoclave before they are finally disposed.  Sharps containers & Trash cans  Sharps containers  Non- are to be used for contaminated the disposal of waste paper or broken glass, other trash needles, used should be scalpel blades, used placed into the glass pipettes, or any regular trash other sharp objects. can.  Reusable materials  Reusable materials that require sterilization should be placed in the autoclave after use. ▪ These include items such as bacterial culture tubes, glass rods, and some instruments that must be sterile for use.  Reusable materials that do not require sterilization may be washed in the sink after use. ▪ These include dissection tools and trays, glassware used for nonhazardous materials, and staining trays.  General rules for handling chemicals  Keep chemical containers closed. Dust and vapor may escape from an open container.  Never use a wrong or an unmarked reagent. If you are unsure about the compound, do not use it. Instead, have it disposed of.  Chemical bottles must not be carried by the neck of the bottle, nor next to your body. Suitable carrying arrangements should be employed, e.g. buckets or trolleys.  When pouring from bottles, the label should always face upward to prevent any spillage from destroying the label.  Never put any chemicals in the bottle other than the one indicated on the label.  Special precautions should be taken when handling concentrated acids. Dilution of acids should be performed by pouring the acid into water and stirring continuously.  In some cases, chemicals may be washed down the sink, but in other cases, they must be handled differently.  The Importance of hand washing  Good hand washing technique is an important practice when conducting laboratory exercises, especially in the field of microbiology.  During the lab, your hands may be in contact with infectious organisms, irritating chemicals, stains, and other potentially hazardous materials.  Your hands may also be a source of contamination, leading to poor results.  Therefore, you should wash your hands prior to the lab exercise, after the lab exercise, and any other time your hands have been in contact with potential hazards.  Steps for Correct Hand Washing Technique  According to the Centers for Disease Control, these are the proper steps to wash your hands thoroughly Remove all jewelry, including Wet your hands with clean, items on your wrists. running water and then apply soap. Steps for Correct Hand Washing Technique Rub your hands together to Continue rubbing your hands for at least 20 make a lather. seconds. For a good timing device, sing or hum the “Happy Birthday” song twice. Be sure to scrub the backs of your hands, between your fingers, and under your nails. Steps for Correct Hand Washing Technique Rinse your hands well under Dry your hands using a clean running water. towel or air drying. Metric systems  Units of measurements  The SI system is the form of measurement typically used by scientists.  The United States is the only industrialized nation that has not adapted the SI system. They still use the English system. All Measurement systems have standards. Standards are exact quantities that everyone agrees to use as a basis of comparison  Units of measurements  In the English system you have to remember so many numbers...  12 inches in a foot  3 feet in a yard  5,280 feet in a mile  16 ounces in a pound  4 quarts to a gallon  In the SI System you only have to remember one number. ▪ The SI System is based on the number 10.  Units of measurements The SI System uses the following prefixes  This system works with any SI measurement.  It is the same system regardless if you are measuring length, mass, or volume.  All measurements need a number and a unit! Micro 1/1000000 Units of measurements References  NIH: Safe Laboratory Practices & Procedures. https://ors.od.nih.gov/sr/dohs/safety/laboratory/Pages/student_goodlab.aspx  CDC: School Chemistry Laboratory Safety Guide (2006). https://www.cdc.gov/niosh/docs/2007-107/pdfs/2007-107.pdf Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Microscopy: types and principles  Learning outcomes  Recognize the principles and uses of different types of microscopes  Identify the parts of compound light microscope and understand their function  Find out the total magnification (objective and ocular lenses)  Understand and follow the rules of microscope use  Focus the compound light microscope at low power and high power magnifications  Define: Field of view (at low and high power), Longitudinal & cross sections and Resolution Quizlet definition: Quizlet definition: https://quizlet.com/527647934/biotechniques_microscopy-flash-cards/  Types of microscopes  Biological objects can be very small to see ❑ Light microscope them with the naked eye. Accordingly scientists ▪ Compound light microscope developed microscopes to view them. ▪ Binocular dissecting microscope (stereomicroscope)  Microscopes come in many different types. ▪ Phase contrast microscope ❑ Fluorescent Microscope ▪ Confocal microscope ❑ Electron microscope ▪ Transmission electron microscope (TEM) ▪ Scanning electron microscope (SEM)  Light microscope  The simplest form of light microscope consists of a single glass lens mounted in a metal frame – a magnifying glass.  The specimen requires very little preparation.  Focusing of the region of interest is achieved by moving the lens and the specimen relative to one another.  The source of light is usually the Sun or ambient indoor light.  The detector is the human eye.  The recording device is a hand drawing.  Compound light microscope  All modern light microscopes are made up of more than one glass lens in combination. The major components are the condenser lens, the objective lens and the eyepiece lens, and, such instruments are therefore called compound microscopes. 1. Light from a lamp is focused at the specimen by a glass condenser lens. 2. The specimen is mounted on a glass slide with a coverslip placed on top. 3. The image is magnified with an objective lens (glass lens). 4. It is projected onto a detector with the eyepiece lens. 5. The detector can be the eye or a digital camera. The parts of the Microscope and their Function Compound light microscope  Lenses and their magnifications OBJECTIVE LENS POWER OF POWER OF TOTAL OCULAR LENS OBJECTIVE LENS MAGNIFICATION SCANING POWER 10X 4X 40 LOW POWER 10X 10X 100 HIGH POWER 10X 40X 400 OIL IMMERSION 10X 100X 1000 Scanning Lower High Oil Total magnification = ocular x objective power power immersion objective objective objective Field of view  Field of view is the area of the slide that you see when you look through a microscope's eyepiece. It is a circle. HP Field of view (40 x) LP Field of view (10x)  Microscope immersion oil  Typically you don’t need the immersion oil with a lower magnification lens. You will need immersion oil when using a higher magnification lens. Microscope immersion oil is a transparent oil that has a unique  Be sure that the lens is optical character and viscosity for use an oil lens. in microscopy, especially in higher magnification.  Stereomicroscope  It is used for the observation of the surfaces of large specimens, when 3D information is required, for micromanipulation and dissections.  Example: routine observation of whole organisms, for example for screening through vials of fruit flies or dissecting a specimen.  A wide range of objectives and eyepieces are available for different applications.  An external light source at different angles serves to add contrast or shadow relief to the images. A research-grade stereomicroscope. The light source is from the side, which can give a shadow effect to the specimen; in this example a vial of fruit flies. The large objective lens above the specimen can be rotated to zoom the image.  Phase contrast microscope  It is used to enhance the contrast of images of transparent and colorless specimens, e.g. viewing unstained cells growing in tissue culture.  It enables visualization of cells and cell components that would be difficult to see using an ordinary light microscope.  The method images differences in the refractive index of cellular structures. ▪ Light that passes through thicker parts of the cell is held up relative to the light that passes through thinner parts of the cytoplasm.  It requires a specialized phase condenser and phase objective lenses.  Fluorescent microscope  Fluorescence microscopy is currently the most widely used contrast technique since it  gives the ability to achieve highly specific labelling of cellular compartments.  gives superior signal to noise ratios, typically white (fluorescent) on a black (non-fluorescent) background.  The most commonly used fluorescence technique is called epifluorescence light microscopy, where ‘epi’ simply means ‘from above’. Here the light source comes from above the sample, and the objective lens acts as both the condenser and the objective lens.  Fluorescent microscope  The light source is the UV into the red wavelengths: ▪ High-pressure mercury or xenon vapour lamp ▪ Lasers ▪ LED sources  This specific wavelength of light is used to excite a fluorescent molecule or fluorophore in the A fluorophore is an organic specimen. molecule with the ability to absorb light at a particular  Light of longer wavelength from the excitation of the fluorophore wavelength and then emit it is then imaged. This is achieved in the fluorescence microscope at a higher wavelength using combinations of filters that are specific for the excitation and emission characteristics of the fluorophore of interest.  Fluorescent proteins & stains  Red fluorescent protein (RFP)  Green fluorescent protein (GFP)  DAPI (4′,6-diamidino-2- phenylindole) is a fluorescent stain that binds strongly to adenine–thymine-rich regions in DNA Fluorescent image of mobile skin cells (fibroblasts) Immunofluorescence  Confocal microscopes Laser scanning confocal microscopes (LSCM)  Optical sections are produced in the laser scanning confocal microscope by scanning the specimen point by point with a laser beam focused in the specimen, and using a spatial filter, usually a pinhole (or a slit), to remove unwanted fluorescence from above and below the focal plane of interest.  The power of the confocal approach lies in the ability to image structures at discrete levels within an intact biological specimen.  Electron microscope  Uses a beam of electrons that is magnified and focused on the object by means of electron magnetics.  Can view much smaller objects compared to light microscopes, with far greater detail. ▪ Transmission electron microscope (TEM) - analogous to the compound light microscope ▪ Scanning electron microscope (SEM) - analogous to dissecting light microscope  Extensive specimen preparation is required for EM analysis, and for this reason there can be issues of interpreting the images because of artifacts from specimen preparation. Transmission electron microscope Scanning electron microscope Microscope use  Rules for microscope use  Keep both eyes open while using the microscope and do not touch the eyepiece with your eye lashes.  The lowest power objective (scanning) should be in the position both at the beginning and end of the microscope use.  Do not clean lenses with regular paper tissue/wipes.  Do not tilt the microscope when viewing.  Do not remove parts of the microscope.  To locate small objects in slide, first find them with the naked eye or under low power.  Focusing the microscope 1. Always begin focusing with the scanning power objective 4x. 2. With coarse adjustment knob lower the stage. 3. Place a slide on the stage and stabilize it with a clip. 4. While looking through the eye piece with both eyes, slowly raise the stage using the coarse adjustment knob until the object comes into view. 5. Use the fine adjustment knob to sharpen the focus if necessary.  Focusing the microscope Compound light microscopes are parfocal, meaning once an object is In focus with the low power field, it should be almost in focus with the higher power. 1. Always find the object under the low power field before viewing it with the high power field. 2. Make sure the object is centered in the middle field. 3. Move the objective lens to the high power objective. You should hear a “click” sound (note: Parfocal microscope objectives will not hit normal slides when changing the objective if the lowest objective was initially used to focus). 4. If any adjustment is needed, use the fine adjustment knob only. Field of view  Field of view is the area of the slide that you see when you look through a microscope's eyepiece. It is a circle. HP Field of view (40 x) LP Field of view (10x) Low-power field diameter (LPD)  Use the 10X (low power) objective.  Use a clear ruler to measure the field diameter in mm.  Convert from mm to micrometer.  This will be your LPD (always expressed in micrometer).  You can calculate the size of a single cell which is equal to: Diameter of the field of view Number of cells that fit LPF = 2 mm (measure by ruler) = 2000 micrometer 1 cell = 2000/ 5 = 400 micrometers e.g. 2 mm diameter  Longitudinal section vs cross section Longitudinal section: cut through the long axis of an organ Transverse section (cross section): a cut along a horizontal plane, dividing the body or organ into superior and inferior parts  Resolution  The resolution achieved by a lens is a measure of its ability to distinguish between two objects in the specimen.  The shorter the wavelengths of illuminating light the higher the resolving power of the microscope.  The limit of resolution for a microscope that uses visible light is about 300nm with a dry lens (in air) and 200nm with an oil immersion lens.  By using ultraviolet light (UV) as a light source the resolution can be improved to 100 nm because of the shorter wavelength of the light (200–300nm). References  The Different Types of Microscopes – A Comprehensive Guide. https://www.microscopeclub.com/types-of-microscopes/  How to Use Microscope Immersion Oil to Get Higher Resolution Images. https://rsscience.com/how-to-use-microscope-immersion-oil-to-get-higher-resolution- images/ Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Acids & Bases  Learning outcomes  Distinguish between atoms and elements.  Distinguish between ionic and covalent bonds.  Define isotope and summarize its application in both medicine and biology.  Describe the properties of water.  Summarize the structure of the pH scale and the importance of buffers to biological systems.  Describe the four classes of organic molecules found in cells.  From atoms to molecules  Matter - anything that has mass and takes up space.  Elements - basic building blocks of matter; cannot be broken down by chemical means.  Over 90% of the human body is made up of only four elements: carbon (C), nitrogen (N), oxygen (O) and hydrogen (H). Carbon (C)  Atom-the basic structural unit of an element. It is the smallest unit of an element that retains the chemical properties of that element.  Atoms consist of three primary particles: protons, neutrons, electrons.  From atoms to molecules  Molecules - atoms bonded together. ▪ Can be made of the same atom or different atoms. That is, O2 , H2O.  Compounds—molecules made of different atoms. ▪ That is, H2O (not O2 ).  Two types of bonds join atoms: ionic bonds and covalent bonds.  Ionic bonding  Atoms are most stable when their outer electron shell, the valence shell, is full.  During an ionic reaction, atoms donate or take on electrons to fill their valence shell.  This results in the formation of positive or negative ions (charged particles). Ionic bond - the attraction between a positive and negative ion. Formation of an ionic bond  Covalent bonding  Each atom contributes one electron to the shared pair.  The electrons spend time in the valence shells of both atoms.  Double covalent bonds share two pairs of electrons; triple covalent bonds share three pairs.  Depicted by one, two, or three straight lines. Covalent bonds - atoms share electrons to fill their valence shells. Covalent bonds  Hydrogen bonds Polarity is a description of how different the electrical poles of a molecule are.  Water is a polar molecule.  Electrons spend more time around the oxygen than the hydrogens, creating a partial negative charge.  Hydrogen bond - attraction between a slightly positive hydrogen to a slightly negative oxygen or nitrogen. ▪ Relatively weak bonds. ▪ Depicted by dotted lines. Hydrogen bonds Water is a Solvent  It dissolves many substances.  Solution—water with dissolved solutes.  Salts dissociate or separate when dissolved in water, facilitating chemical reactions.  Water is a solvent  Polar molecules attract water, so are hydrophilic.  Nonpolar molecules DO NOT attract water, so are hydrophobic. ▪ In nonpolar covalent bonds, the electrons are shared equally (no partial charges).  Acids  Acids are substances that dissociate in water, releasing hydrogen ions (H+). ▪ That is, hydrochloric acid (HCl) is produced by the stomach and aids in digestion.  Bases  Bases are substances that take up hydrogen ions (H+)or release hydroxide ions (OH−). ▪ That is, sodium hydroxide (NaOH) is a strong base.  The pH scale  The pH scale is a measure of acidity or basicity (alkalinity) of a solution. ▪ Ranges from 0 (very acidic) to 14 (very basic). ▪ 7 is neutral - hydrogen ion (H+)concentration is equal to hydroxide (OH−) concentration. ▪ A pH below 7 is acidic (H+ is greater than OH−) and above 7 is basic (OH− is greater than H+). ▪ The concentration of hydrogen ions between each pH number changes by a factor of 10.  Buffers  Buffer - a solution that resists changes in pH when acids or bases are added to it. ▪ Important within the body or in the ecosystem, where pH values need to stay within a narrow range.  Buffers act to establish an equilibrium between a conjugate acid/base pair. Buffers consist of either: ▪ a weak acid and its salt (conjugate base) ▪ a weak base and its salt (conjugate acid)  Buffers can be destroyed by the addition of too much acid or base. Buffer capacity - a measure of the ability of a solution to resist large changes in pH when a strong acid or strong base is added. Biomolecules  Molecules of life  The four major organic molecules in the body: ▪ Carbohydrates. ▪ Lipids. ▪ Proteins. ▪ Nucleic acids.  Each is composed of subunits.  Molecules of life  Dehydration reaction - a type of synthesis  Hydrolysis reaction - the addition of water to chemical reaction that removes water, break macromolecules into their subunits. linking subunits together into macromolecules (large molecules).  Carbohydrates  Used as an energy source.  Simple carbohydrates (monosaccharides) are made of a single sugar molecule: ▪ Glucose. ▪ Fructose. ▪ Galactose.  Carbohydrates  Disaccharides are made of two monosaccharides joined by a dehydration reaction. Sucrose is table sugar Lactose is milk sugar  Carbohydrates  Polysaccharides (complex carbohydrates) - long polymers of glucose subunits. Starch Glycogen Cellulose  Proteins  Amino acids - the subunits of proteins.  Each consists of an amino group, a carboxyl group and an R group. Each amino acid differs in its R group.  Proteins  Polypeptide - three or more amino acids linked together.  Peptide bond - the polar covalent bond between two amino acids.  Shape of proteins  A protein’s 3-dimensional shape is closely linked to its function.  Denaturation - the change in the shape of a protein  Caused by extreme heat or pH.  Disrupts the protein’s function.  Lipids  Lipids are biomolecules that are soluble in nonpolar solvents  Found in the form of: ▪ Triglycerides (fats and oils). ▪ Phospholipids. ▪ Steroids.  Triglycerides  Made of one glycerol and three fatty acids.  Come in two forms: fats and oils ▪ Fats: usually animal origin & solid at room temperature. ▪ Oils: usually plant origin & liquid at room temperature.  Triglycerides are hydrophobic. Emulsifiers are molecules that surround triglycerides and disperse, or emulsify, them.  Phospholipids  Structure is similar to a triglyceride but one fatty acid is replaced by a polar phosphate group.  Have a polar, hydrophilic ‘head’ and nonpolar, hydrophobic ‘tails’.  Are the primary components of plasma membranes.  Steroids - precursor to other steroids like the sex hormones  Lipids made of four fused carbon rings.  Each type differs in the functional group attached to the rings.  Nucleic acids  Polymers of nucleotides composed of: ▪ a phosphate. ▪ a 5 carbon sugar. ▪ a nitrogenous base.  Nucleic acids, concluded: ▪ Deoxyribonucleic acid (DNA) ▪ Ribonucleic acid (RNA) Help regulate enzyme action Nucleotides are commonly identified by ▪ Components of coenzymes their base, since that is the only component ▪ Adenosine triphosphate (ATP) that differs within a nucleic acid. DNA compared to RNA Complementary base pairing Adenine always binds to thymine Cytosine always binds to guanine Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Basic Molecular Techniques  Learning outcomes  Define molecular biology methods and techniques.  Explain DNA cloning techniques.  Describe DNA and plasmid extraction.  Describe the CRISPR-Cas9 system.  Describe DNA synthesis and DNA microarray.  Explain the different types of DNA mutation.  Molecular biology methods  Molecular biology methods used to study the molecular basis of biological activity.  The essence of cell chemistry to isolate a particular cellular component and then analyze its chemical structure and activity.  Methods most commonly used to explore cells, their characteristics & processes: ▪ Nucleic acid methods. ▪ Protein methods. ▪ Immunostaining methods. Method is the approach or pathway (a process ). Technique is a man made strategy or tactic (practical aspects).  Molecular biology techniques  DNA cloning:  Gel electrophoresis:  Molecular hybridization: ▪ Cut & paste DNA ▪ Various types ▪ Southern blot ▪ DNA or RNA isolation ▪ Northern blot ▪ Ligation  Reading and writing DNA: ▪ Western blot ▪ Bacterial transformation or transfection ▪ Chromosome integration ▪ DNA sequencing ▪ Expression cloning ▪ DNA synthesis  Cell culture: ▪ Cellular screening  Polymerase Chain Reaction (PCR):  Rewriting DNA: mutations ▪ DNA polymerase DNA dependent ▪ Random mutagenesis  Arrays: ▪ PCR dynamics ▪ Point mutation ▪ DNA array ▪ PCR types ▪ Chromosome mutation ▪ Protein array ▪ CRISPR/Cas9  DNA cloning overview The inserted DNA is reproduced along with the vector Cut and paste DNA  Join two molecules togeth er: ▪ insert : usually smaller ▪ vector : has origin of replication  Two types of vectors are most Circular double stranded plasmid DNA Recombinant vector commonly used: (Cloning vector) ▪ E. coli plasmid vectors. ▪ bacteriophage λ vectors.  Cut and paste DNA: plasmids  Plasmids are circular, self-replicating, double-stranded DNA (dsDNA) m olecules th at are separate from a cell’s chromosomal DNA.  Plasmids have b een engineere d to optimize their use as vectors (reduced length ≈3kb; contain only the essential nucleotide sequences): ▪ restriction sites ▪ marker genes for selection and/or screening ▪ origin of replication  Plasmid vectors replicate along with their host cells (low, medium or high copy number). Cut and paste DNA: plasmids  A DNA fragment of a few base pairs up to ≈20 kb can be inserted into a plasmid vector.  Like the host-cell chromosomal DNA, pDNA is duplicated before every cell division. Cut and paste DNA: euk expression vectors  Eukaryote: viruses ▪ restriction sites ▪ virus genes ▪ terminal repeats  Cut and paste DNA: restriction  Restriction sites are specific 4- to 8-bp sequences that are recognized by restriction endonucleases (restrictases).  Many restriction sites are short inverted repeat sequences.  Restriction enzyme type 2 cut DNA: ▪ highly specific (type II endonucleases) ▪ leaves blunt or sticky ends Cut and paste DNA: restriction Cut and paste DNA: restriction  Cut and paste DNA: restriction ▪ DNA ligase catalyzes formation of 3’ → 5′ covalent bonds between the short fragments. ▪ Restriction fragments are covalently ligated together: ▪ Restriction fragments with complementary “sticky ends” are ligated easily. ▪ Blunt-end ligation requires a higher DNA concentration than ligation of sticky ends. DNA transfer into cells  Chromosome integration  Integration of the target genes into the host chromosome. ▪ preferable strategy to overcome the drawbacks of plasmid-based overexpression. ▪ plasmid-free stable mutants. ▪ possible in bacteria but usually necessary in eukaryotes.  Integrase is the enzyme that splices the viral DNA into a cellular chromosome. Chromosome integration Transposon- mediated gene transposition Homologous recombination a single Site-specific recombination is an exchange that crossover between a targeting gene occurs between pairs of defined sequences and a homologous DNA fragment on a (target sites). This process is mediated by a chromosome. The whole plasmid specific recombinase that can be expressed via sequence is integrated. a helper plasmid.  Cellular screening  Both vectors are derived from natural plasmids, but both have been genetically modified for convenient use as vectors. ▪ The plasmid pBR322 is simpler in structure. ▪ The pUC plasmid is a more advanced vector, whose structure allows direct visual selection of colonies containing vectors with donor DNA inserts. β-galactosidase is a protein encoded by the lacZ gene  DNA extraction  DNA extraction (isolation) is a method to purify DNA by using physical and/or chemical methods from a sample separating DNA from cell membranes, proteins, and other cellular components. ▪ The aim is to tak e only DNA f rom the whole cell extract.  The basic steps of DNA extraction: ▪ Lysis: the cell and the nucleus are broken open to release the DNA (mechanical disruption or chemical lysis with detergents, e.g. Proteinase K ). ▪ Precipitation: separation of DNA from the cellular debris (Na+ ions neutralize the negative charges on the DNA molecules & they are precipitated from aqueous solutions in ethanol or isopropanol). Silica membrane ▪ Purification: rinsed with alcohol to remove any remaining unwanted material. ▪ Elution: with either the elution buffer or with water.  Extraction of plasmids  Purification of plasmid DNA from bacterial DNA is based on the differential denaturation of chromosomal and plasmid DNA using alkaline lysis in order to separate the two. ▪ Neutralization with potassium acetate allows only the covalently closed plasmid DNA to reanneal and to stay solubilized. ▪ Chromosomal DNA and proteins removed by centrifugation. Dirty minipreps DNA cloning overview Crispr/Cas9  CRISPR-Cas9 was adapted from a naturally occurring genome editing system in bacteria. ▪ A Cas enzyme for cutting the target sequence. ▪ A single guide RNA (sgRNA), which binds to the target sequence of 20bp (PAM sequence).  CRISPR/Cas9 creates specific double-strand breaks at the target locus. These corrections result in two types of genome modifications: DNA synthesis  Commercial synthesis  Because artificial gene synthesis does not require template DNA, it is theoretically possible to make a completely synthetic DNA molecule with no limits on the nucleotide sequence or size.  Gene synthesis - $0.09/bp  DNA array Expression profiles  A microarray is a laboratory tool used to detect the expression of thousands of genes at the same time. ▪ Probe is DNA molecules of variable length on a solid support (oligo chip). ▪ Sample is labeled DNA or RNA that will bind to the probes.  The principle behind microarrays is that complementary sequences will bind to each other.  DNA array  DNA microarrays are microscope slides that are printed with thousands of tiny spots in defined positions, with each spot containing a known DNA sequence or gene. Clustering of expression profiles defines breast cancer cell line subtypes. https://doi.org/10.1371/journal.pone.0006146  Mutations and causes Mutations are alterations in DNA sequences that result in changes in the structure of a gene.  Spontaneous: ▪ At low frequency owing to 1) the chemical instability of purine and pyrimidine bases and 2) to errors during DNA replication.  Induced: ▪ Exposure of an organism to certain environmental factors may increase the frequency of spontaneous mutations. ✓ Chemical mutagens induce point mutations ✓ Ionizing radiation gives rise to large chromosomal abnormalities.  Main types of mutations  In biological systems that are capable of reproduction, we must first focus on whether mutations are heritable (offspring, and further descendants).  By the cell type where mutations occur, they are classified as: ▪ Germline mutations occur in gametes. ▪ Somatic mutations occur in other body cells.  By the size of the involved region, mutations can be classified as: ▪ Point mutations ▪ Chromosomal mutations nuclear DNA mutations ▪ Copy number variation (CNV) vs mtDNA mutations  Point mutations  Substitution: One base is incorrectly added during r eplication and replaces the pair in the corresponding position on the complementary strand.  Insertion: One or more extra nucleotides are inserted into replicating DNA, often resulting in a frameshift.  Deletion: One or more nucleotides is/are "skipped" during replication or otherwise excised, often resulting in a frameshift.  Point mutations  Missense (nonsynonymous): ▪ A single nucleotide resulting in a codon that codes for a different amino acid.  Nonsense: ▪ A single nucleotide resulting in a premature stop codon.  Synonymous: ▪ A single nucleotide that changes a codon to an amino acid with similar properties e.g. Lysine to Arginine.  Silent: ▪ A single nucleotide that does not alter amino acid sequences e.g. GCT, GCC, GCA and GCG all code for alanine.  Neutral: ▪ A single nucleotide that does not have any harmful or beneficial effect on the organism, it usually occurs at noncoding DNA regions.  Point mutations Mutations can also be categorized on the basis of the function:  The loss-of-function mutations cause a decrease or a loss of the gene product or the activity of the gene product.  The gain-of-function mutations cause an increase in the amount of gene product or its activity, and sometimes create a new property, leading to a toxic product responsible for a pathological effect.  Chromosomal mutations  Deletion: A region of a chromosome is lost, resulting in the absence of all the genes in that area.  Duplication: A region of a chromosome is repeated, resulting in an increase in dosage from the genes in that region.  Inversion: One region of a chromosome is flipped and reinserted.  Insertion: A region of a chromosome is cut from one chromosome and inserted into another. ▪ interchromosomal insertion - another non-homologous chromosome ▪ intrachromosomal insertion - a different region of the same chromosome  Translocation: A breakage in two chromosomes and each of the broken pieces reunites with another chromosome. Copy number variation (CNV)  Gene amplification: The number of tandem copies of a locus is increased.  Expanding trinucleotide repeat: The normal number of repeated trinucleotide sequences is expanded. Because CNVs change the structure of the genome, such mutations, together with inversions and translocations, are collectively classified as forms of genome structural variation.  Mutation hotspots  Genetic mutations are influenced by sequence context, structure, and genomic features.  Some areas of the genome tend to be more prone to mutations than others. These regions in a genome exhibit elevated rates of recombination relative to a neutral expectation.  These "hot spots" are often a result of the DNA sequence itself being more accessible to mutagens. Hot spots include areas of the genome with highly repetitive sequences, such as trinucleotide repeats.  Mitochondrial DNA mutations  In some cases, inherited changes in mitochondrial DNA can cause problems with growth, development, and function of the body's systems.  mtDNA mutations disrupt the mitochondria's ability to generate energy efficiently for cells. Human mitochondrial DNA (mtDNA) References  Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 7.1, DNA Cloning with Plasmid Vectors.  Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. Site-Specific Recombination.  Griffiths AJF, Gelbart WM, Miller JH, et al. Modern Genetic Analysis. New York: W. H. Freeman; 1999. Cloning a Specific Gene.  Govindarajan R, Duraiyan J, Kaliyappan K, Palanisamy M. Microarray and its applications. J Pharm Bioallied Sci. 2012;4(Suppl 2):S310-S312. doi:10.4103/0975-7406.100283  Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 8.1, Mutations: Types and Causes.  Clancy, S. (2008) Genetic mutation. Nature Education 1(1):187  Mahdieh N, Rabbani B. An overview of mutation detection methods in genetic disorders. Iran J Pediatr. 2013;23(4):375-388.  Eichler, E. E. (2008) Copy Number Variation and Human Disease. Nature Education 1(3):1  Loewe, L. (2008) Genetic mutation. Nature Education 1(1):113 Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] PCR & Electrophoresis  Learning outcomes  Explain PCR technique.  Identify some Polymerases.  Describe TA cloning.  Describe Gel electrophoresis.  Polymerase Chain Reaction (PCR)  Polymerase chain reaction (PCR) is a laboratory technique used to amplify DNA sequences. ▪ The method involves using short DNA sequences called primers to select the portion of the genome to be amplified. ▪ PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. ▪ The technique can produce a billion copies of the target sequence in just a few hours.  Polymerase Chain Reaction (PCR)  DNA-dependent DNA polymerase copies DNA into DNA DNA polymerase ▪ adds dNTP to a 3’OH end of an existing strand. 3 DNA-dependent DNA polymerase Reverse transcriptase RNA-dependent DNA-dependent 1 4 DNA polymerase RNA polymerase RNA-dependent 2 RNA polymerase RNA polymerase RNA replicase Components of PCR  DNA template - the sample DNA that contains the target sequence.  Primers - short pieces of single-stranded DNA that are complementary to the target sequence. The polymerase begins synthesizing new DNA.  Nucleotides (dNTPs or deoxynucleotide triphosphates) - single units of the bases A, T, G, and C, which are essentially "building blocks" for new DNA strands.  DNA polymerase - type of enzyme that synthesizes new strands of DNA complementary to the target sequence.  DNA polymerase buffer - a buffer that creates optimal conditions for the polymerase to work.  Water to adjust final concentrations.  Components of PCR  DNA template: 104-107 molecules (50-100 ng of gDNA OR 10-50ng of plasmid DNA).  Primers: 0.1–1 μM (degenerated primers 0.3–1 μM).  Nucleotides: 50 μM of each of the four nucleotides (20-200 μM).  DNA polymerase: 0.5-2.5 units per 50 μl.  DNA polymerase buffer - X buffer.  Sterile water to adjust final folume. Total volume: 15μL  Why are 2 primers needed for PCR?  Two primers, forward primer and reverse primer are designed to flank the target region for amplification. ▪ The forward primer binds to the template DNA. ▪ The reverse primer binds to the other complementary (coding) strand. Plus “+” Sense Coding Complementary Non-template Template Complementary Minus “-” Antisense Non-coding Orientation of primers  PCR primer design  Primers should generally have the following  Primers should g enerally have the following properties: properties: ▪ Length of 18-24 bases ▪ Length of 18-30 bases ▪ 40-60% G/C content ▪ 40-60% G/C content ▪ End with G/C (GC Clamp). ▪ Start and end with 1-2 G/C pairs ▪ Melting temperature (Tm) of 65-75°C ▪ Melting temperature (Tm) of 50-60°C ▪ Primer pairs should have a Tm within 5°C of each other ▪ Primer pairs should have a Tm within 5°C of each other ▪ Avoid regions of secondary structure, GC-rich and AT- ▪ Primer pairs should not have complementary regions rich domains. ▪ Avoid runs of 4 or more of one base, or dinucleotide repeats, e.g. ACCCC or ATATATAT  Note: If you will be including a restriction site at the 5’ end of your primer, note that a 3-6 bp "clamp" should be added upstream in order for the enzyme to cleave efficiently. PCR principle  PCR is based on three simple steps required for any DNA synthesis reaction: (1)denaturation of the template into single strands (2)annealing of primers to each original strand for new strand synthesis (3) extension of the new DNA strands from the primers. PCR profile At its optimal temperature (72°C), Taq polymerase incorporates nucleotides at a rate of 1-2 kb per minute. PCR program  Polymerases commercially available  DNA polymerase I. ▪ The commercial form is extracted from E. coli. ▪ Its 5' to 3' DNA polymerase activity requires a template. ▪ it also has 3' to 5' and 5' to 3' exonuclease activity. ▪ This enzyme is used to synthesize DNA from a single-strand DNA template at 37ºC.  The Klenow Fragment of DNA polymerase I. ▪ It is a fragment of DNA polymerase I obtained by limited proteolysis. ▪ The 5' to 3' exonuclease activity is removed. ▪ The 5' to 3' polymerase and the 3' to 5’ exonuclease activities are preserved.  Polymerases commercially available  Taq DNA Polymerase. ▪ This is a thermostable enzyme isolated from Thermus aquaticus. ▪ It is used for PCR amplification of DNA fragments up to 5 kb in length. ▪ It is also used for DNA labelling and sequencing. ▪ It is ideal for TA cloning. It has a non-template dependent activity that adds a single adenosine to the 3' ends. The downside of Pfu is its speed  Pfu DNA polymerase. which is slower than that of Taq. ▪ It derives from the hyperthermophilic archae Pyrococcus furiosus. ▪ It has 3' to 5' exonuclease activity and a high proofreading efficiency ▪ It lacks 5' to 3' exonuclease activity. ▪ It is used for high-fidelity PCR and primer-extension reactions and the generation of blunt-end amplification products.  Polymerases commercially available Commercially purified Taq polymerase At its optimal  Phusion. doesn't have a proof-reading domain, so temperature (72°C), Taq it has a higher error rate. polymerase i ncorporates ▪ It has extreme fidelity and high speed. nucleotides at a rate of ▪ It allows high product yields with minimal enzyme concentrations. 1-2 kilobases per minute. ▪ It is capable of amplifying long templates. Phusion requires 15-30  Terminal transferase. seconds per kb. This ▪ This is a mammalian enzyme expressed in lymphocytes. means 4 kb per minute. ▪ It catalyzes deoxynucleotide addition to a free 3'-OH end without the need for a template. The choice of deoxynucleotide added is made randomly. The base composition of the synthesized polydeoxynucleotide depends on the base concentrations in the incubation medium. ▪ It is an example of a DNA polymerase that does not require a primer. ▪ It is used to generate DNA blunt ends and for labelling of DNA 3' ends TA cloning  Taq polymerase has non-template dependent activity which preferentially adds a single adenosine to the 3'-ends of a double stranded DNA molecule, and thus most of the molecules PCR amplified by Taq polymerase possess single 3'-A overhangs.  TA cloning vector was designed so that when linearized, it has single 5′-T overhangs at each end.  TA cloning is one of the simplest and most efficient methods for the cloning of PCR products.  Gel electrophoresis  Gel electrophoresis is a laboratory method used to separate mixtures of molecules (DNA, RNA, or proteins) according to their molecular size. ▪ DNA is negatively charged. When an electric current is applied to the gel, DNA will migrate towards the positively charged electrode. ▪ The molecules to be separated are pushed by an electrical field through a gel that contains small pores. DNA is negatively charged  DNA is negatively charged because of the presence of phosphate groups in nucleotides.  The phosphate backbone of DNA is negatively charged, which is due to the presence of bonds created between the phosphorus and oxygen atoms.  Each phosphate group contains one negatively charged oxygen atom.  Agarose gel  Agarose is a group of natural polysaccharides derived from seaweed (red algae). It’s a derivative of agar.  Agarose can be dissolved in boiling water and a gel is formed after cooling this solution below 45 °C as a result of extensive hydrogen- bonding between the agarose chains.  The pore size of agarose gels depends on the agarose content.  6 % agarose have an average pore size of approximately 30 nm, whereas 4 and 2 % agarose beads have a pore size of 70 and 150 nm, respectively.  Agarose with even larger diffusion pores are used in gel electrophoresis to allow the passage of very large DNA molecules.  Polyacrylamide gels  3 major differences between agarose & polyacrylamide gels, which lead to their distinct uses in the research lab. ▪ Toxicity: agarose is considered entirely non-toxic, whereas polyacrylamide powders and gels are considered moderately hazardous and require protection during handling. ▪ Molecular complexity: Agarose is complex and has wide gaps between the many differently-sized molecules that make up the gel matrix. Polyacrylamide is made up of only one large molecular type. ▪ Gel preparation: Agarose is poured horizontally, and polyacrylamide is poured vertically.  degrading the nucleic acids. Running buffers  The function of the running buffer is to allow nucleic acids to move through the agarose matrix. Therefore, the agarose gel must be submerged in the buffer.  The running buffer maintains the pH and ion concentration during electrophoresis. The pH stays within the appropriate range which is important because the changes in pH affect the net charge of the nucleic acids.  The running buffer contains EDTA (ethylenediaminetetraacetic acid) prevents nuclease from degrading the nucleic acids. TAE works better for cloning, because TBE contains borate.  The main running buffers are: TAE and TBE: Borate in TBE is an inhibitor for many enzymes, such as ligase. ▪ TAE is based on acetic acid. TAE produces a better separation of TAE works better for performing DNA ▪ TBE is based on boric acid. larger fragments (>3kb). extraction from agarose gel. TBE is suitable for obtaining a higher TBE has a higher buffering capacity - resolution of smaller fragments (